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Patent 2395398 Summary

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(12) Patent Application: (11) CA 2395398
(54) English Title: NUCLEIC ACIDS, PROTEINS, AND ANTIBODIES
(54) French Title: ACIDES NUCLEIQUES, PROTEINES, ET ANTICORPS
Status: Withdrawn
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/12 (2006.01)
  • C07H 21/02 (2006.01)
  • C07H 21/04 (2006.01)
  • C07K 5/00 (2006.01)
  • C07K 14/00 (2006.01)
  • C07K 14/47 (2006.01)
  • C07K 14/705 (2006.01)
  • C07K 16/18 (2006.01)
  • C12N 1/20 (2006.01)
  • C12N 5/00 (2006.01)
  • C12N 9/00 (2006.01)
  • C12N 9/64 (2006.01)
  • C12N 15/63 (2006.01)
  • C12P 21/06 (2006.01)
  • A61K 38/00 (2006.01)
  • A61K 48/00 (2006.01)
  • C12Q 1/68 (2006.01)
(72) Inventors :
  • ROSEN, CRAIG A. (United States of America)
  • BARASH, STEVEN C. (United States of America)
  • RUBEN, STEVEN M. (United States of America)
(73) Owners :
  • ROSEN, CRAIG A. (Not Available)
  • BARASH, STEVEN C. (Not Available)
  • RUBEN, STEVEN M. (Not Available)
(71) Applicants :
  • HUMAN GENOME SCIENCES, INC. (United States of America)
(74) Agent: MBM INTELLECTUAL PROPERTY LAW LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2001-01-17
(87) Open to Public Inspection: 2001-08-02
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2001/001358
(87) International Publication Number: WO2001/055163
(85) National Entry: 2002-06-20

(30) Application Priority Data:
Application No. Country/Territory Date
60/179,065 United States of America 2000-01-31
60/214,886 United States of America 2000-06-28
60/246,613 United States of America 2000-11-08
60/246,609 United States of America 2000-11-08
60/246,478 United States of America 2000-11-08
60/246,524 United States of America 2000-11-08
60/246,523 United States of America 2000-11-08
60/249,299 United States of America 2000-11-17
60/249,210 United States of America 2000-11-17
60/249,216 United States of America 2000-11-17
60/249,217 United States of America 2000-11-17
60/249,211 United States of America 2000-11-17
60/215,135 United States of America 2000-06-30
60/249,215 United States of America 2000-11-17
60/249,218 United States of America 2000-11-17
60/249,208 United States of America 2000-11-17
60/249,213 United States of America 2000-11-17
60/249,212 United States of America 2000-11-17
60/249,207 United States of America 2000-11-17
60/249,245 United States of America 2000-11-17
60/249,244 United States of America 2000-11-17
60/249,297 United States of America 2000-11-17
60/249,214 United States of America 2000-11-17
60/216,647 United States of America 2000-07-07
60/249,264 United States of America 2000-11-17
60/249,209 United States of America 2000-11-17
60/249,300 United States of America 2000-11-17
60/249,265 United States of America 2000-11-17
60/250,391 United States of America 2000-12-01
60/250,160 United States of America 2000-12-01
60/256,719 United States of America 2000-12-05
60/251,030 United States of America 2000-12-05
60/251,988 United States of America 2000-12-05
60/251,479 United States of America 2000-12-06
60/216,880 United States of America 2000-07-07
60/251,869 United States of America 2000-12-08
60/251,856 United States of America 2000-12-08
60/251,868 United States of America 2000-12-08
60/251,990 United States of America 2000-12-08
60/251,989 United States of America 2000-12-08
60/254,097 United States of America 2000-12-11
60/259,678 United States of America 2001-01-05
60/217,487 United States of America 2000-07-11
60/217,496 United States of America 2000-07-11
60/218,290 United States of America 2000-07-14
60/220,963 United States of America 2000-07-26
60/220,964 United States of America 2000-07-26
60/225,757 United States of America 2000-08-14
60/180,628 United States of America 2000-02-04
60/225,270 United States of America 2000-08-14
60/225,447 United States of America 2000-08-14
60/225,267 United States of America 2000-08-14
60/225,758 United States of America 2000-08-14
60/225,268 United States of America 2000-08-14
60/224,518 United States of America 2000-08-14
60/224,519 United States of America 2000-08-14
60/225,759 United States of America 2000-08-14
60/225,213 United States of America 2000-08-14
60/225,266 United States of America 2000-08-14
60/184,664 United States of America 2000-02-24
60/225,214 United States of America 2000-08-14
60/226,279 United States of America 2000-08-18
60/226,868 United States of America 2000-08-22
60/227,182 United States of America 2000-08-22
60/226,681 United States of America 2000-08-22
60/227,009 United States of America 2000-08-23
60/228,924 United States of America 2000-08-30
60/229,344 United States of America 2000-09-01
60/229,343 United States of America 2000-09-01
60/229,287 United States of America 2000-09-01
60/186,350 United States of America 2000-03-02
60/229,345 United States of America 2000-09-01
60/229,513 United States of America 2000-09-05
60/229,509 United States of America 2000-09-05
60/230,438 United States of America 2000-09-06
60/230,437 United States of America 2000-09-06
60/231,413 United States of America 2000-09-08
60/232,080 United States of America 2000-09-08
60/231,414 United States of America 2000-09-08
60/231,244 United States of America 2000-09-08
60/232,081 United States of America 2000-09-08
60/189,874 United States of America 2000-03-16
60/231,242 United States of America 2000-09-08
60/231,243 United States of America 2000-09-08
60/231,968 United States of America 2000-09-12
60/232,401 United States of America 2000-09-14
60/232,399 United States of America 2000-09-14
60/232,400 United States of America 2000-09-14
60/232,397 United States of America 2000-09-14
60/233,063 United States of America 2000-09-14
60/233,064 United States of America 2000-09-14
60/233,065 United States of America 2000-09-14
60/190,076 United States of America 2000-03-17
60/232,398 United States of America 2000-09-14
60/234,223 United States of America 2000-09-21
60/234,274 United States of America 2000-09-21
60/234,997 United States of America 2000-09-25
60/234,998 United States of America 2000-09-25
60/235,484 United States of America 2000-09-26
60/235,834 United States of America 2000-09-27
60/235,836 United States of America 2000-09-27
60/236,369 United States of America 2000-09-29
60/236,327 United States of America 2000-09-29
60/198,123 United States of America 2000-04-18
60/236,370 United States of America 2000-09-29
60/236,368 United States of America 2000-09-29
60/236,367 United States of America 2000-09-29
60/237,039 United States of America 2000-10-02
60/237,038 United States of America 2000-10-02
60/237,040 United States of America 2000-10-02
60/237,037 United States of America 2000-10-02
60/236,802 United States of America 2000-10-02
60/239,937 United States of America 2000-10-13
60/239,935 United States of America 2000-10-13
60/205,515 United States of America 2000-05-19
60/241,785 United States of America 2000-10-20
60/241,809 United States of America 2000-10-20
60/240,960 United States of America 2000-10-20
60/241,787 United States of America 2000-10-20
60/241,808 United States of America 2000-10-20
60/241,221 United States of America 2000-10-20
60/241,786 United States of America 2000-10-20
60/241,826 United States of America 2000-10-20
60/244,617 United States of America 2000-11-01
60/246,474 United States of America 2000-11-08
60/209,467 United States of America 2000-06-07
60/246,532 United States of America 2000-11-08
60/246,476 United States of America 2000-11-08
60/246,526 United States of America 2000-11-08
60/246,475 United States of America 2000-11-08
60/246,525 United States of America 2000-11-08
60/246,528 United States of America 2000-11-08
60/246,527 United States of America 2000-11-08
60/246,477 United States of America 2000-11-08
60/246,611 United States of America 2000-11-08
60/246,610 United States of America 2000-11-08

Abstracts

English Abstract




The present invention relates to novel proteins. More specifically, isolated
nucleic acid molecules are provided encoding novel polypeptides. Novel
polypeptides and antibodies that bind to these polypeptides are provided. Also
provided are vectors, host cells, and recombinant and synthetic methods for
producing human polynucleotides and/or polypeptides, and antibodies. The
invention further relates to diagnostic and therapeutic methods useful for
diagnosing, treating, preventing and/or prognosing disorders related to these
novel polypeptides. The invention further relates to screening methods for
identifying agonists and antagonists of polynucleotides and polypeptides of
the invention. The present invention further relates to methods and/or
compositions for inhibiting or enhancing the production and function of the
polypeptides of the present invention.


French Abstract

La présente invention concerne de nouvelles protéines et, plus particulièrement, des molécules d'acide nucléique isolées codant de nouveaux polypeptides, ainsi que de nouveaux polypeptides et des anticorps qui se lient à ces polypeptides. L'invention concerne également des vecteurs, des cellules hôtes, et des méthodes recombinantes et synthétiques de production de polynucléotides et/ou de polypeptides humains et des anticorps. Elle concerne des méthodes diagnostiques et thérapeutiques servant à diagnostiquer, traiter, prévenir et/ou pronostiquer des troubles liés à ces nouveaux polypeptides. Elle concerne en outre des méthodes de criblage permettant d'identifier des agonistes et des antagonistes des polynucléotides et polypeptides de l'invention. L'invention concerne enfin des méthodes et/ou des compositions pouvant inhiber ou améliorer la production et la fonction des polypeptides de l'invention.

Claims

Note: Claims are shown in the official language in which they were submitted.





What Is Claimed Is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence at least 95% identical to a sequence selected from the
group consisting
of:
(a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of
the
cDNA sequence contained in Clone ID NO:Z, which is hybridizable to SEQ ID
NO:X;
(b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a
polypeptide fragment encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z,
which is hybridizable to SEQ ID NO:X;
(c) a polynucleotide encoding a polypeptide fragment of a polypeptide encoded
by
SEQ ID NO:X or a polypeptide fragment encoded by the cDNA sequence contained
in
cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X;
(d) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a
polypeptide domain encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z,
which is hybridizable to SEQ ID NO:X;
(e) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a
polypeptide epitope encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z,
which is hybridizable to SEQ ID NO:X;
(f) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA
sequence
contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X, having
biological activity;
(g) a polynucleotide which is a variant of SEQ ID NO:X;
(h) a polynucleotide which is an allelic variant of SEQ ID NO:X;
(i) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y;
(j) a polynucleotide capable of hybridizing under stringent conditions to any
one of
the polynucleotides specified in (a)-(i), wherein said polynucleotide does not
hybridize under
stringent conditions to a nucleic acid molecule having a nucleotide sequence
of only A
residues or of only T residues.

2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide
fragment comprises a nucleotide sequence encoding a protein.



676




3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide
fragment comprises a nucleotide sequence encoding the sequence identified as
SEQ ID NO:Y
or the polypeptide encoded by the cDNA sequence contained in cDNA Clone ID
NO:Z,
which is hybridizable to SEQ ID NO:X.

4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide
fragment comprises the entire nucleotide sequence of SEQ ID NO:X or the cDNA
sequence
contained in cDNA Clone ID NO:Z, which is hybridizable to SEQ ID NO:X.

5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide
sequence comprises sequential nucleotide deletions from either the C-terminus
or the N-
terminus.

6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide
sequence comprises sequential nucleotide deletions from either the C-terminus
or the N-
terminus.

7. A recombinant vector comprising the isolated nucleic acid molecule of claim
1.

8. A method of making a recombinant host cell comprising the isolated nucleic
acid molecule of claim 1.

9. A recombinant host cell produced by the method of claim 8.

10. The recombinant host cell of claim 9 comprising vector sequences.

11. An isolated polypeptide comprising an amino acid sequence at least 90%
identical to a sequence selected from the group consisting of:
(a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in
cDNA Clone ID NO:Z;
(b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence contained in
cDNA Clone ID NO:Z, having biological activity;



677




(c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence contained in
cDNA Clone ID NO:Z;
(d) a polypeptide epitope of SEQ ID NO:Y or the encoded sequence contained in
cDNA Clone ID NO:Z;
(e) a full length protein of SEQ ID NO:Y or the encoded sequence contained in
cDNA
Clone ID NO:Z;
(f) a variant of SEQ ID NO:Y;
(g) an allelic variant of SEQ ID NO:Y; or
(h) a species homologue of the SEQ ID NO:Y.

12. The isolated polypeptide of claim 11, wherein the full length protein
comprises sequential amino acid deletions from either the C-terminus or the N-
terminus.

13. An isolated antibody that binds specifically to the isolated polypeptide
of
claim 11.

14. A recombinant host cell that expresses the isolated polypeptide of claim
11.

15. A method of making an isolated polypeptide comprising:
(a) culturing the recombinant host cell of claim 14 under conditions such that
said
polypeptide is expressed; and
(b) recovering said polypeptide.

16. The polypeptide produced by claim 15.

17. A method for preventing, treating, or ameliorating a medical condition,
comprising administering to a mammalian subject a therapeutically effective
amount of the
polynucleotide of claim 1.

18. A method of diagnosing a pathological condition or a susceptibility to a
pathological condition in a subject comprising:
(a) determining the presence or absence of a mutation in the polynucleotide of
claim
1; and
678




(b) diagnosing a pathological condition or a susceptibility to a pathological
condition
based on the presence or absence of said mutation.

19. A method of diagnosing a pathological condition or a susceptibility to a
pathological condition in a subject comprising:

(a) determining the presence or amount of expression of the polypeptide of
claim 11
in a biological sample; and

(b) diagnosing a pathological condition or a susceptibility to a pathological
condition
based on the presence or amount of expression of the polypeptide.

20. A method for identifying a binding partner to the polypeptide of claim 11
comprising:
(a) contacting the polypeptide of claim 11 with a binding partner; and

(b) determining whether the binding partner effects an activity of the
polypeptide.

21. The gene corresponding to the cDNA sequence of SEQ ID NO:Y.

22. A method of identifying an activity in a biological assay, wherein the
method
comprises:
(a) expressing SEQ ID NO:X in a cell;

(b) isolating the supernatant;

(c) detecting an activity in a biological assay; and
identifying the protein in the supernatant having the activity.

23. The product produced by the method of claim 20.

24. A method for preventing, treating, or ameliorating a medical condition,
comprising administering to a mammalian subject a therapeutically effective
amount of the
polypeptide of claim 11.

679

Description

Note: Descriptions are shown in the official language in which they were submitted.





DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
~~ TTENANT LES PAGES 1 A 379
NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des
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NOTE POUR LE TOME / VOLUME NOTE:


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
Nucleic Acids, Proteins, and Antibodies
[1] This application refers to a "Sequence Listing" that is provided only on
electronic
media in computer readable form pursuant to Administrative Instructions
Section 801 (a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and
Administrative Instructions Sections 801 to 806, and is hereby incorporated in
its entirety.
[2] The Sequence Listing is provided as an electronic file (PJZ07 seqList.txt,
1,401,914 bytes in size, created on January 13, 2001) on four identical
compact discs (CD-
R), labeled "COPY 1," "COPY 2," "COPY 3,"-end "CRF." The Sequence Listing
complies
with Annex C of the Administrative Instructions, and may be viewed, for
example, on an
IBM-PC machine running the MS-Windows operating system by using the V viewer
software, version 2000 (see World Wide Web URL: http://www.fileviewer.com).
Field of the ~Tnventiore
[3] The present invention relates to novel proteins. More specifically,
isolated nucleic
acid molecules are provided encoding novel polypeptides. Novel polypeptides
and antibodies
that bind to these polypeptides are provided. Also provided are vectois, host
cells, and
recombinant and synthetic methods for producing human polynucleotides and/or
polypeptides, and antibodies. The invention further relates to diagnostic and
therapeutic
methods useful for diagnosing, treating, preventing and/or prognosing
disorders related to
these novel polypeptides. The invention further relates to screening methods
for identifying
1


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
agonists and antagonists of polynucleotides and polypeptides of the invention.
The present
invention further relates to methods and/or compositions for inhibiting or
enhancing the
production and function of the polypeptides of the,present invention.
Background of the Invention
[4j Neoplastic disease (or cancer) is actually a diverse group of diseases
sharing one
characteristic in common; all neoplastic diseases (cancers) result from the
uncontrolled
proliferation of cells. The human body is composed of many different cell
types, e.g. liver
cells, muscle cells, brain cells, etc. Normally, these cells grow and divide
to produce more
cells only as the body needs them (e.g. to regenerate blood cells or replace
epithelial cells
lining the stomach). Sometimes, however, cells begin to divide unchecked even
though new
cells are not needed. These extra cells accumulate and form a mass of tissue,
called a tumor.
Although each of the over 200 cell types in the body can potentially become
cancerous, some
cell types become cancerous at relatively high rates while many other cell
types rarely
become cancerous.
[5j Tumors are either benign or malignant. Benign tumors are not cancerous;
they can
usually be removed, they do not spread to other parts of the body and, they
rarely threaten
life. Malignant tumors, however, are cancerous. Cells in malignant tumors can
invade and
damage nearby or distant tissues and organs. The spread of cancerous cells is
called
metastasis. Malignant (or metastatic) cells can invade adjacent organs by
proliferating
directly from the primary tumor. Additionally, malignant cells can also
metastasize to distant
organs by breaking away from the primary tumor, entering the bloodstream or
lymphatic
system, and settling down in a new organ or tissue to produce a secondary
tumor. The origin
of secondary tumors is established by comparing cells comprising these tumors
to cells in the
original (primary) tumor.
[6j In conhast to solid organ cancers (such as cancer in the liver, lung, and
brain)
cancer can also develop in blood-forming cells. These cancers are referred to
as leukemias or
lymphomas. Leukemia refers to cancer of blood forming cells such as red blood
cells,
platelets, and plasma cells. Lymphomas are a subset of leukemias, primarily
involving white
blood cells, in which the cancerous cells originated in, or are associated
with, the lymph
system and lymph organs (e.g. T-lymphocytes iri the lymph nodes, spleen, or
thymus).
2


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
[7] In 1999 over 1.1 million people were newly diagnosed with 23 different
types of
cancer. The vast majority of these cases (~75%) involved cancers of the
prostate, breast,
lung, colon, or urinary tract, or non-Hodgkin's lymphoma. Among the most fatal
cancers are
pancreatic, liver, esophageal, lung, stomach, and brain cancers, having up to
96% mortality
rates depending on the specific cancer. In all, some 23 different types of
cancer are expected
to kill over 86,000 people each year.
[8] Most cancers are treated with one or a combination therapies consisting of
surgery,
radiation therapy, chemotherapy, hormone therapy, and/or biological therapy.
These five
therapeutic modes are either local or systemic treatment strategies. Local
treatments affect
cancer cells in the tumor and imediately adjacent areas (for example, surgical
tumor removal
is a local treatment as are most radiation treatments). In contrast, systemic
treatments travel
through the bloodstream, and reach cancer and other cells all over the body.
Chemotherapy,
hormone therapy, and biological therapy are examples of systemic treatments.
(9] Whether systemic or local, it is often difficult or impossible to protect
healthy cells
from the harmful effects of cancer treatment; healthy cells and tissues are
inevitably damaged
in the process of treating the cancerous cells. Damage and disruption of the
normal
functioning of healthy cells and tissues often produces the undesirable side
effects
experienced by patients undergoing cancer treatment.
[10] Recombinant polypeptides and polynucleotides derived from naturally
occurnng
molecules, as well as antibodies specifically targeted to these molecules,
used alone or in
conjunction with other existing therapies, hold great promise as improved
therapeutic agents
for the treatment of neoplastic disorders. Currently, most biological therapy
can be classified
as immunotherapy because these treatments often use naturally occurring
molecules to assist
the body's immune system in fighting the disease or in protecting the body
from side effects
of other cancer treatment(s). Among the most commonly used compounds in
biological
therapies are proteins called cytokines (e.g. interferons, interleukins, and
colony stimulating
factors) and monoclonal antibodies (targeted to particular cancer cells). Side
effects caused
by these commonly used biological therapies range from flu-like symptoms
(chills, fever,
muscle aches, weakness, loss of appetite, nausea, vomiting, and diarrhea) to
rashes, swelling,
easy bruising, or bleeding.
[1l] The discovery of new human neoplastic disease associated polynucleotides,
the
polypeptides encoded by ' them, and antibodies that immunospecifically bind
these
polypeptides, satisfies a need in the art by providing new compositions which
are useful in
3


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
the diagnosis, treatment, prevention and/or prognosis of disorders involving
neoplastic
diseases.
Summary of the Invention
[12] The present invention relates to novel proteins. More specifically,
isolated nucleic
acid molecules are provided encoding novel polypeptides. Novel polypeptides
and antibodies
that bind to these polypeptides are provided. Also provided are vectors, host
cells, and
recombinant and synthetic methods for producing human polynucleotides and/or
polypeptides, and antibodies. The invention further relates to diagnostic and
therapeutic
methods useful for diagnosing, treating, preventing and/or prognosing
disorders related to
these novel polypeptides. The invention further relates to screening methods
for identifying
agonists and antagonists of polynucleotides and polypeptides of the invention.
The present
invention further relates to methods and/or compositions for inhibiting or
enhancing the
production and function of the polypeptides of the present invention.
Detailed Description
Tables
[13] Table 1A summarizes some of the polynucleotides encompassed by the
invention
(including cDNA clones related to the sequences (Clone )D NO:Z), contig
sequences (contig
identifier (Contig >D:) and contig nucleotide sequence identifier (SEQ >D
NO:X)) and further
summarizes certain characteristics of these polynucleotides and the
polypeptides encoded
thereby. The first column provides the gene number in the application for each
clone
identifier. The second column provides a unique clone identifier, "Clone ll~
NO:Z", for a
cDNA clone related to each contig sequence disclosed in Table 1A. The third
column
provides a unique contig identifier, "Contig >D:" for each of the contig
sequences disclosed
in Table 1A. The fourth column provides the sequence identifier, "SEQ m NO:X",
for each
of the contig sequences disclosed in Table 1A. The fifth column, "ORF (From-
To)",
provides the location (i.e., nucleotide position numbers) within the
polynucleotide sequence
of SEQ m NO:X that delineate the preferred open reading frame (ORF) that
encodes the
amino acid sequence shown in the sequence listing and referenced in Table 1A
as SEQ >D
NO:Y (column 6). Column 7 lists residues comprising predicted epitopes
contained in the
4


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y).
Identification of
potential immunogenic regions was performed according to the method of Jameson
and Wolf
(CABIOS, 4; 181-186 (1988)); specifically, , the Genetics Computer Group (GCG)
implementation of this algorithm, embodied in the program PEPT>DESTRUCTURE
(Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wisc.). This
method
returns a measure of the probability that a given residue is found on the
surface of the protein.
Regions where the antigenic index score is greater than 0.9 over at least 6
amino acids are
indicated in Table 1A as "Predicted Epitopes". In particular embodiments,
polypeptides of
the invention comprise, or alternatively consist of, one, two, three, four,
five or more of the
,predicted epitopes described in Table 1A. It will be appreciated that
depending on the
analytical criteria used to predict antigenic determinants, the exact address
of the determinant
may vary slightly. Column 8, "Tissue Distribution" shows the expression
profile of tissue,
cells, and/or cell line libraries which express the polynucleotides of the
invention. The first
number in column 8 (preceding the colon), represents the tissue/cell source
identifier code
corresponding to the key provided in Table 4. Expression of these
polynucleotides was not
observed in the other tissues and/or cell libraries tested. For those
identifier codes in which
the first two letters are not "AR", the second number in column 8 (following
the colon),
represents the number of times a sequence corresponding to the reference
polynucleotide
sequence (e.g., SEQ ID NO:X) was identified in the tissue/cell source. Those
tissue/cell
source identifier codes in which the first two letters are "AR" designate
information
generated using DNA array technology. Utilizing this technology, cDNAs were
amplified by
PCR and then transferred, in duplicate, onto the array. Gene expression was
assayed through
hybridization of first strand cDNA probes to the DNA array. cDNA probes were
generated
from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis
was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse
transcription.
After hybridization, high stringency washing conditions were employed to
remove non-
specific hybrids from the array. The remaining signal, emanating from each
gene target, was
measured using a Phosphorimager. Gene expression was reported as Phosphor
Stimulating
Luminescence (PSL) which reflects the level of phosphor signal generated from
the probe
hybridized to each of the gene targets represented on the array. A local
background signal
subtraction was performed before the total signal generated from each array
was used to
normalize gene expression between the different hybridizations. The value
presented after
"[array code]:" represents the mean of the duplicate values, following
background subtraction
S


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
and probe normalization. One of skill in the art could routinely use this
information to
identify normal and/or diseased tissues) which show a predominant expression
pattern of the
corresponding polynucleotide of the invention or to identify polynucleotides
which show
predominant and/or specific tissue and/or cell expression. Column 9 provides
the
chromosomal location of polynucleotides corresponding to SEQ )D NO:X.
Chromosomal
location was determined by finding exact matches to EST and cDNA sequences
contained in
the NCBI (National Center for Biotechnology Information) UniGene database.
Given a
presumptive chromosomal location, disease locus association was determined by
comparison
with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online
Mendelian
Inheritance in Man, OMIMTM. McKusick-Nathans Institute for Genetic Medicine,
Johns
Hopkins University (Baltimore, MD) and National Center for Biotechnology
Information,
National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim~. If the putative chromosomal location of the
Query
overlaps with the chromosomal location of a Morbid Map entry, an OMIM
identification
number is disclosed in column 10 labeled "OMIM Disease References)". A key to
the
OMIM reference identification numbers is provided in Table 5.
[14] Table 1B summarizes additional polynucleotides encompassed by the
invention
(including cDNA clones related to the sequences (Clone >D NO:Z), contig
sequences (contig
identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)),
and genomic
sequences (SEQ 1D NO:B). The first column provides a unique clone identifier,
"Clone 1D
NO:Z", for a cDNA clone related to each contig sequence. The second column
provides the
sequence identifier, "SEQ 1D NO:X", for each contig sequence. The third column
provides a
unique contig identifier, "Contig )D:" for each contig sequence. The fourth
column, provides
a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the
corresponding row of
the table. The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" for
a fragment of the BAC clone identified in column four of the corresponding row
of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide
position numbers)
within the polynucleotide sequence of SEQ 1D NO:B which delineate certain
polynucleotides
of the invention that are also exemplary members of polynucleotide sequences
that encode
polypeptides of the invention (e.g., polypeptides containing amino acid
sequences encoded
by the polynucleotide sequences delineated in column six, and fragments and
variants
thereof).
6


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
[15] Table 2 summarizes homology and features of some of the polypeptides of
the
invention. The first column provides a unique clone identifier, "Clone ID
NO:Z",
corresponding to a cDNA clone disclosed in Table 1A. The second column
provides the
unique contig identifier, "Contig )D:" corresponding to contigs in Table 1A
and allowing for
correlation with the information in Table 1A. The third column provides the
sequence
identifier, "SEQ >D NO:X", for the contig polynucleotide sequence. The fourth
column
provides the analysis method by which the homology/identity disclosed in the
Table was
determined. Comparisons were made between polypeptides encoded by the
polynucleotides
of the invention and either a non-redundant protein database (herein referred
to as "NR"), or
a database of protein families (herein referred to as "PFAM") as further
described below.
The fifth column provides a description of the PF~dVI/NR hit having a
significant match to a
polypeptide of the invention. Column six provides the accession number of the
PFAM/NR
hit disclosed in the fifth column. Column seven, "Score/Percent Identity",
provides a quality
score or the percent identity, of the hit disclosed in columns five and six.
Columns 8 and 9,
"NT From" and "NT To" respectively, delineate the polynucleotides in "SEQ ID
NO:X" that
encode a polypeptide having a significant match to the PFAM/NR database as
disclosed in
the fifth and sixth columns. In specific embodiments polypeptides of the
invention comprise,
or alternatively consist of, an amino acid sequence encoded by a
polynucleotide in SEQ ID
NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
[16] Table 3 provides polynucleotide sequences that may be disclaimed
according to
certain embodiments of the invention. The first column provides a unique clone
identifier,
"Clone >D", for a cDNA clone related to contig sequences disclosed in Table
1A. The
second column provides the sequence identifier, "SEQ m NO:X", for contig
sequences
disclosed in Table 1A. The third column provides the unique contig identifier,
"Contig ll~:",
for contigs disclosed in Table 1A. The fourth column provides a unique integer
'a' where 'a'
is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and
the fifth
column provides a unique integer 'b' where 'b' is any integer between 15 and
the final
nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ )D NO:X, and where b is greater than or equal to a + 14.
For each of
the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be
substituted
into the general formula of a-b, and used to describe polynucleotides which
may be
preferably excluded from the invention. In certain embodiments, preferably
excluded from
the invention are at least one, two, three, four, five, ten, or more of the
polynucleotide
7


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
sequences) having the accession numbers) disclosed in the sixth column of this
Table
(including for example, published sequence in connection with a particular BAC
clone). In
further embodiments, preferably excluded from the invention are the specific
polynucleotide
sequences) contained in the clones corresponding to at least one, two, three,
four, five, ten,
or more of the available material having the accession numbers identified in
the' sixth column
of this Table (including for example, the actual sequence contained in an
identified BAC
clone).
[17] Table 4 provides a key to the tissue/cell source identifier code
disclosed in Table
1A, column 8. Column 1 provides the tissue/cell source identifier code
disclosed in Table 1A,
Column 8. Columns 2-S provide a description of the tissue or cell source.
Codes
corresponding to diseased tissues are indicated in column 6 with the word
"disease". The use
of the word "disease" in column 6 is non-limiting. The tissue or cell source
may be specific
(e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a
normal portion
of a diseased organ). Furthermore, tissues and/or cells lacking the "disease"
designation may
still be derived from sources directly or indirectly involved in a disease
state or disorder, and
therefore may have a further utility in that disease state or disorder. In
numerous cases where
the tissue/cell source is a library, column 7 identifies the vector used to
generate the library.
[18] . Table S provides a key to the OMIM reference identification numbers
disclosed in
Table 1A, column 10. OMIM reference identification numbers (Column 1) were
derived
from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man,
OM1M.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University
(Baltimore,
MD) and National Center for Biotechnology Information, National Library of
Medicine,
(Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim~.
Column 2 provides diseases associated with the cytologic band disclosed in
Table 1A,
column 9, as determined using the Morbid Map database.
[19] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation
numbers of deposits made with the ATCC in connection with the present
application.
[20] Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers
and
vector information relating to these cDNA libraries.
[21] Table 8 provides a physical characterization of clones encompassed by the
invention. The first column provides the unique clone identifier, "Clone ID
NO:Z", for
certain cDNA clones of the invention, as described in Table 1A. The second
column provides
the size of the cDNA insert contained in the corresponding cDNA clone.
8


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
Definitions
[22] The following definitions are provided to facilitate understanding of
certain terms
used throughout this specification.
[23] In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is altered "by
the hand of man" from its natural state. For example, an isolated
polynucleotide could be
part of a vector or a composition of matter, or could be contained within a
cell, and still be
"isolated" because that vector, composition of matter, or particular cell is
not the original
environment of the polynucleotide. The term "isolated" does not refer to
genomic or cDNA
libraries, whole cell total or mRNA preparations, genomic DNA preparations
(including
those separated by electrophoresis and transferred onto blots), sheared whole
cell genomic
DNA preparations or other compositions where the art demonstrates no
distinguishing
features of the polynucleotide/sequences of the present invention.
[24J As used herein, a "polynucleotide" refers to a molecule having a nucleic
acid
sequence encoding SEQ >D NO:Y or a fragment or variant thereof; a nucleic acid
sequence
contained in SEQ )D NO:X (as described in column 3 of Table 1A) or the
complement
thereof; a cDNA sequence contained in Clone >D NO:Z (as described in column 2
of Table
1 A and contained within a library deposited with the ATCC); a nucleotide
sequence encoding
the polypeptide encoded by a nucleotide sequence in SEQ 1D NO:B as defined in
column 6
of Table 1B or a fragment or variant thereof; or a nucleotide coding sequence
in SEQ m
NO:B as defined in column 6 of Table 1B or the complement thereof. For
example, the
polynucleotide can contain the nucleotide sequence of the full length cDNA
sequence,
including the 5' and 3' untranslated sequences, the coding region, as well as
fragments,
epitopes, domains, and variants of the nucleic acid sequence. Moreover, as
used herein, a
"polypeptide" refers to a molecule having an amino acid sequence encoded by a
polynucleotide of the invention as broadly defined (obviously excluding poly-
Phenylalanine
or poly-Lysine peptide sequences which result from translation of a polyA tail
of a sequence
corresponding to a cDNA).
(25J In the present invention, "SEQ >D NO:X" was often generated by
overlapping
sequences contained in multiple clones (contig analysis). A representative
clone containing
all or most of the sequence for SEQ 1D NO:X is deposited at Human Genome
Sciences, Inc.
9


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
(HGS) in a catalogued and archived library. As shown, for example, in column 2
of Table
1A, each clone is identified by a cDNA Clone )D (identifier generally referred
to herein as
Clone 1D NO:Z). Each .Clone m is unique to an individual clone and the Clone
>D is all the
information needed to retrieve a given clone from the HGS library.
Furthermore, certain
clones disclosed in this application have been deposited with the ATCC on
October 5, 2000,
having the ATCC designation numbers PTA 2574 and PTA 2575; and on January 5,
2001,
having the depositor reference numbers TS-1, TS-2, AC-1, and AC-2. In addition
to the
individual cDNA clone deposits, most of the cDNA libraries from which the
clones were
derived were deposited at the American Type Culture Collection (hereinafter
"ATCC").
Table 7 provides a list of the deposited cDNA libraries. One can use the Clone
>D NO:Z to
determine the library source by reference to Tables 6 and 7. Table 7 lists the
deposited
cDNA libraries by narrie and links each library to an ATCC Deposit. Library
names contain
four characters, for example, "HTWE." The name of a cDNA clone (Clone m)
isolated from
that library begins with the same four characters, for example "HTWEP07". As
mentioned
below, Table 1A correlates the Clone m names with SEQ m NO:X. Thus, starting
with an
SEQ 1D NO:X, one can use Tables 1, 6 and 7 to determine the corresponding
Clone m,
which library it came from and which ATCC deposit the library is contained in.
Furthermore,
it is possible to retrieve a given cDNA clone from the source library by
techniques known in
the art and described elsewhere herein. The ATCC is located at 10801
University Boulevard,
Manassas, Virginia 20110-2209, USA. The ATCC deposits were made pursuant to
the terms
of the Budapest Treaty on the international recognition of the deposit of
microorganisms for
the purposes of patent procedure.
[26] In specific embodiments, the polynucleotides of the invention are at
least 15, at
least 30, at least 50, at least 100, at least 125, at least 500, or at least
1000 continuous
nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15
kb, 10 kb, 7.5kb, 5
kb, 2.5 kb; 2.0 kb, or 1 kb, in length. In a further embodiment,
polynucleotides of the
invention comprise a portion of the coding sequences, as disclosed herein, but
do not
comprise all or a portion of any intron. In another embodiment, the
polynucleotides
comprising coding sequences do not contain coding sequences of a genomic
flanking gene
(i.e., 5' or 3' to the gene of interest in the genome). In other embodiments,
the
polynucleotides of the invention do not contain the coding sequence of more
than 1000, 500,
250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
[27] A "polynucleotide" of the present invention also includes those
polynucleotides
capable of hybridizing, under stringent hybridization conditions, to sequences
contained in
SEQ m NO:X, or the complement thereof (e.g., the complement of any one, two,
three, four,
or more of the polynucleotide fragments described herein), the polynucleotide
sequence
delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or
cDNA sequences
contained in Clone ID NO:Z (e.g., the complement of any one, two, three, four,
or more of
the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones
deposited
with the ATCC, described herein), and/or the polynucleotide sequence
delineated in column
6 of Table 1B or the complement thereof. "Stringent hybridization conditions"
refers to an
overnight incubation at 42 degree C in a solution comprising 50% formamide, 5x
SSC (750
mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x
Denhardt's
solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm
DNA,
followed by washing the filters in O.lx SSC at about 65 degree C.
[28] Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides
of the present invention at lower stringency hybridization conditions. Changes
in the
stringency of hybridization and signal detection are primarily accomplished
through the
manipulation of formamide concentration (lower percentages of formamide result
in lowered
stringency); salt conditions, or temperature. For example, lower stringency
conditions
include an overnight incubation at 37 degree C in a solution comprising 6X
SSPE (20X SSPE
= 3M NaCI; 0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100
ug/ml
salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE, 0.1%
SDS.
In addition, to achieve even lower stringency, washes performed following
stringent
hybridization can be done at higher salt concentrations (e.g. 5X SSC).
[29] Note that variations in the above conditions may be accomplished through
the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in
hybridization experiments. Typical blocking reagents include Denhardt's
reagent, BLOTTO,
heparin, denatured salmon sperm DNA, and commercially available proprietary
formulations.
The inclusion of specific blocking reagents may require modification of the
hybridization
conditions described above, due to problems with compatibility.
[30] Of course, a polynucleotide which hybridizes only to polyA+ sequences
(such as
any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a
complementary stretch of T (or U) residues, would not be included in the
definition of
"polynucleotide," since such a polynucleotide would hybridize to any nucleic
acid molecule
11


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
containing a poly (A) stretch or the complement thereof (e.g., practically any
double-stranded
cDNA clone generated using oligo dT as a primer).
[31] The polynucleotide of the present. invention can be composed of any
polyribonucleotide or polydeoxribonucleotide; which may be unmodified RNA or
DNA or
modified RNA or DNA. For example, polynucleotides can be composed of single-
and
double-stranded DNA, DNA that .is a mixture of single- and double-stranded
regions, single-
and double-stranded RNA, and RNA that is mixture of single- and double-
stranded regions,
hybrid molecules comprising DNA and RNA that may be single-stranded or, more
typically,
double-stranded or a mixture of single- and double-stranded regions. In
addition, the
polynucleotide can be composed of triple-stranded regions comprising RNA or
DNA or both
RNA and DNA. A polynucleotide may also contain one or more modified bases or
DNA or
RNA backbones modified for stability or for other reasons. "Modified" bases
include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications can
be made to DNA and RNA; thus, "polynucleotide" embraces chemically,
enzymatically, or
metabolically modified forms.
[32] The polypeptide of the present invention can be composed of amino acids
joined to
each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres, and may
contain amino acids other than the 20 gene-encoded amino acids. The
polypeptides may be
modified by either natural processes, such as posttranslational processing, or
by chemical
modification techniques which are well known in the art. Such modifications
are well
described in basic texts and in more detailed monographs, as well as in a
voluminous
research literature. Modifications can occur anywhere in a polypeptide,
including the peptide
backbone, the amino acid side-chains and the amino or carboxyl termini. It
will be
appreciated that the same type of modification may be present in the same or
varying degrees
at several sites in a given polypeptide. Also, a given polypeptide may contain
many types of
modifications. Polypeptides may be branched, for example, as a result of
ubiquitination, and
they may be cyclic, with or without branching. Cyclic, branched, and branched
cyclic
polypeptides may result from posttranslation natural processes or may be made
by synthetic
methods. Modifications include acetylation, acylation, ADP-ribosylation,
amidation,
covalent attachment of flavin, covalent attachment of a heme moiety, covalent
attachment of
a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid
derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine, formation
12


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor
formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation,
proteolytic
processing, phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-
RNA mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.
Enzymol. 182:626-
646 ( 1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 ( 1992)).
[33] "SEQ B7 NO:X" refers to a polynucleotide sequence described, for example,
in
Tables lAor 2, while "SEQ >D NO:Y" refers to a polypeptide sequence described
in column
6 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 4
of Table 1A.
The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF)
encoded
by polynucleotide SEQ ID NO:X. "Clone ID NO:Z" refers to a cDNA clone
described in
column 2 of Table 1A.
[34] "A polypeptide having functional activity" refers to a polypeptide
capable of
displaying one or more known functional activities associated with a full-
length (complete)
protein. Such functional activities include, but are not limited to,
biological activity,
antigenicity [ability to bind (or compete with a polypeptide for binding) to
an anti-
polypeptide antibody], immunogenicity (ability to generate antibody which
binds to a
specific polypeptide of the invention), ability to form multimers with
polypeptides of the
invention, and ability to bind to a receptor or ligand for a polypeptide.
(35] The polypeptides of the invention can be assayed for functional activity
(e.g.
biological activity) using or routinely modifying assays known in the art, as
well as assays
described herein. Specifically, one of skill in the art may routinely assay
neoplastic disease
associated polypeptides (including fragments and variants) of the invention
for activity using
assays as described in Examples 21-62 and 65.
[36] "A polypeptide having biological activity" refers to a polypeptide
exhibiting
activity similar to, but not necessarily identical to, an activity of a
polypeptide of the present
invention, including mature forms, as measured in a particular biological
assay, with or
without dose dependency. In the case where dose dependency does exist, it need
not be
identical to that of the polypeptide, but rather substantially similar to the
dose-dependence in
a given activity as compared to the polypeptide of the present invention
(i.e., the candidate
13


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
polypeptide will exhibit greater activity or not more than about 25-fold less
and, preferably,
not more than about tenfold less activity, and most preferably, not more than
about three-fold
less activity relative to the polypeptide of the present invention).
[37] Table 1A summarizes some of the polynucleotides encompassed by the
invention
(including contig sequences (SEQ m NO:X) and clones (Clone m NO:Z) and further
summarizes certain characteristics of these polynucleotides and the
polypeptides encoded
thereby.
14


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
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CA 02395398 2002-06-20
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CA 02395398 2002-06-20
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[38] The first column in Table 1A provides the gene number in the application
responding to the clone identifier. The second column in Table iA provides a
unique
"Clone >D NO:Z" for a cDNA clone related to each contig sequence disclosed in
Table 1A.
This clone ID references the cDNA clone which contains at least the S' most
sequence of the
assembled contig and at least a portion of SEQ 11.7 NO'X was determined by
directly
sequencing the referenced clone. The reference clone may have more sequence
than
described in the sequence listing or the clone may have less. In the vast
majority of cases,
however, the clone is believed to encode a full-length polypeptide. In the
case where a clone
is not full-length, a full-length cDNA can be obtained by methods described
elsewhere
herein.
[39] The third column in Table 1A provides a unique "Contig >D" identification
for
each contig sequence. The fourth column provides the "SEQ ID NO:" identifier
for each of
the contig polynucleotide sequences disclosed in Table 1A. The fifth column,
"ORF (From-
To)", provides the location (i.e., nucleotide position numbers) within the
polynucleotide
sequence "SEQ ~ NO:X" that delineate the preferred open reading frame (ORF)
shown in
the sequence listing and referenced in Table 1A, column 6, as SEQ m NO:Y.
Where the
nucleotide position number "To" is lower than the nucleotide position number
"From", the
preferred ORF is the reverse complement of the referenced polynucleotide
sequence.
(40] The sixth column in Table 1A provides the corresponding SEQ >D NO:Y for
the
polypeptide sequence encoded by the preferred ORF delineated in column 5. In
one
embodiment, the invention provides an amino acid sequence comprising, or
alternatively
consisting of, a polypeptide encoded by the portion of SEQ 117 NO:X delineated
by "ORF
(From-To)". Also provided are polynucleotides encoding such amino acid
sequences and the
complementary strand thereto.
(41] Column 7 in Table 1A lists residues comprising epitopes contained in the
polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using
the
algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The
Jameson-Wolf
antigenic analysis was performed using the computer program PROTEAN (Version
3.11 for
the Power Macintosh, DNASTAR, Inc., 1228 South Park Street Madison, WI). In
specific
embodiments, polypeptides of the invention comprise, or alternatively consist
of, at least one,
two, three, four, five or more of the predicted epitopes as described in Table
lA. It will be
appreciated that depending on the analytical criteria used to predict
antigenic determinants,
the exact address of the determinant may vary slightly.
114


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
[42] Column 8 in Table 1 A provides an expression profile and library code:
count for
h of the contig sequences (SEQ >D NO:X) disclosed in Table 1A, which can
routinely be
combined with the information provided in Table 4 and used to determine the
tissues, cells,
and/or cell line libraries which predominantly express the polynucleotides of
the invention.
The first number in column 8 (preceding the colon), represents the tissue/cell
source
identifier code corresponding to the code and description provided in Table 4.
For those
identifier codes in which the first two letters are not "AR", the second
number in column 8
(following the colon) represents the number of times a sequence corresponding
to the
reference polynucleotide sequence was identified in the tissue/cell source.
Those tissue/cell
source identifier codes in which the first two letters are "AR" designate
information
generated using DNA array technology. Utilizing this technology, cDNAs were
amplified by
PCR and then transferred, in duplicate, onto the array. Gene expression was
assayed through
hybridization of first strand cDNA probes to the DNA array. cDNA probes were
generated
from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis
was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse
transcription.
After hybridization, high stringency washing conditions were employed to
remove non-
specific hybrids from the array. The remaining signal, emanating from each
gene target, was
measured using a Phosphorimager. Gene expression was reported as Phosphor
Stimulating
Luminescence (PSL) which reflects the level of phosphor signal generated from
the probe
hybridized to each of the gene targets represented on the array. A local
background signal
subtraction was performed before the total signal generated from each array
was used to
normalize gene expression between the different hybridizations. The value
presented after
"[array code]:" represents the mean of the duplicate values, following
background subtraction
and probe normalization. One of skill in the art could routinely use this
information to
identify normal and/or diseased tissues) which show a predominant expression
pattern of the
corresponding polynucleotide of the invention or to identify polynucleotides
which show
predominant and/or specific tissue and/or cell expression.
[43] Column 9 in Table 1A provides a chromosomal map location for certain
polynucleotides of the invention. Chromosomal location was determined by
finding exact
matches to EST and cDNA sequences contained in the NCBI (National Center for
Biotechnology Information) UniGene database. Each sequence in the UniGene
database is
assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a cluster are
believed to be
derived from a single gene. Chromosomal mapping data is often available for
one or more
115


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
sequences) in a UniGene cluster; this data (if consistent) is then applied to
the cluster as a
ale. Thus, it is possible to infer the chromosomal location of a new
polynucleotide
sequence by determining its identity with a mapped UniGene cluster.
[44] A modified version of the computer program BLASTN (Altshul et al., J.
Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet: 3:266-272 (1993))
was used to
search the UniGene database for EST or cDNA sequences that contain exact or
near-exact
matches to a polynucleotide sequence of the invention (the 'Query'). A
sequence from the
UniGene database (the 'Subject') was said to be an exact match if it contained
a segment of
50 nucleotides in length such that 48 of those nucleotides were in the same
order as found in
the Query sequence. If all of the matches that met this criteria were in the
same UniGene
cluster, and mapping data was available for this cluster, it is indicated in
Table 1A under the
heading "Cytologic Band". Where a cluster had been further localized to a
distinct cytologic
band, that band is disclosed; where no banding information was available, but
the gene had
been localized to a single chromosome, the chromosome is disclosed.
[45] Once a presumptive chromosomal location was determined for a
polynucleotide of
the invention, an associated disease locus was identified by comparison with a
database of
diseases which have been experimentally associated with genetic loci. The
database used was
the Morbid Map, derived from OMIMTM (supra). If the putative chromosomal
location of a
polynucleotide of the invention (Query sequence) was associated with a disease
in the
Morbid Map database, an OMIM reference identification number was noted in
column 10,
Table 1 A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference
identification numbers (column 1), and provides a description of the
associated disease in
Column 2.
116


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TABLE 1 B
Clone SEQ ID CONTIG BAC ID: SEQ ID EXON
ID A ~


NO:Z NO:X ID: NO:B From-To


HMSKF13 12 708207 AC023891 579 1-32


159-474


659-1496


2796-2957


3160-3603


5493-5676


8391-8506


8716-8880


8891-9116


9977-10703


11326-


12477


HMSKF13 12 708207 AC023891 580 1-169


HLHCT68 14 764745 AC010344 581 1-121


2985-3815


4282-4423


HLHCT68 14 764745 AC008496 582 1-96


2321-2645


5406-5532


5545-5688


6055-6205


9065-9895


10362-


10503


HLHCT68 14 764745 AC010344 583 1-144


HLHCT68 14 764745 AC010344 584 1-2396


HLHCT68 14 764745 AC008496 585 1-1684


HSSJM44 18 871067 AC026352 586 1-2314


HSSJM44 18 871067 AC068755 587 1-2311


HSSJM44 18 871067 AC069443 588 1-2314


HSSJM44 18 871067 AC026352 589 1-430


HSSJM44 18 871067 AC068755 590 1-430


HSSJM44 18 871067 AC069443 591 1-430


HLMD095 26 928344 AC020641 592 1-591


627-2046


HCEMY90 27 932927 AC024242 593 1-274


1243-1357


1994-2270


HCEMY90 27 932927 AF214633 594 1-274


1243-1357


1994-2270


HCEMY90 27 932927 AC024242 595 1-232


HCEMY90 27 932927 AF214633 596 1-130


HBGQN46 28 945370 AF038458 597 1-630


1311-1416


117


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WO 01/55163 PCT/USO1/01358
2481-4022


4952-5252


63 70-6479


7623-8269


HRODF07 30 952426 AC023402 598 1-336


HRODF07 30 952426 AC011597 599 1-336


HE8DL23 41 693641 AL135999 600 1-63


405-942


1196-1502


2152-6417


6659-6755


7033-7385


7481-7535


7647-8163


8230-8492


8590-9909


10114-


10360


10420-


10783


10970-


11960


12018-


13492


14130-


14528


14563-


15789


HE8DL23 41 693641 AL135999 601 1-410


HACCH94 53 847143 AL161458 602 1-1140


HACCH94 53 847143 AL161458 603 1-90


5811-6312


HE8AM04 65 871156 AL031774 604 1-50


653-725


1150-1308


1333-1455


2269-2559


3501-3804


5007-5125


6175-6302


7148-7335


7528-7651


9888-10170


10252-


10407


11442-


11707


14428-


16375


118


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WO 01/55163 PCT/USO1/01358
16690-


20376


20668-


21969


21986-


26710


28521-


28830


28970-


29123


29902-


30040


32282-


32637


33088-


33347


33353-


33578


34231-


34609


34931-


35398


39684-


40049


41912-


42363


42740-


43003


43570-


44745


46120-


46437


48252-


48374


48882-


48976


49047-


49398


50471-


50599


50820-


50928


S 1281-


51572


51740-


51954


56361-


56634


56904-


57144


119


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WO 01/55163 PCT/USO1/01358
HE8AM04 65 871156 AL138825 605 1-50


653-725


1169-1487


2288-2578


3503-3806


5009-5127


6177-6304


7150-7337


7531-7654


9900-10172


10257-


10332


HE8AM04 65 871156 AL138825 606 1-181


HE8AM04 65 871156 AL031774 607 1-117


HE8AM04 65 871156 AL138825 608 1-309


HIBEF26 66 871533 299716 609 1-54


4060-4540


4625-4819


8967-9094


9489-9787


9908-10459


10603-


10741


12152-


12665


13046-


13140


13458-


13707


14554-


14649


1 S 645-


15745


16067-


16249


17272-


17324


17601-


17925


18108-


18949


19292-


21535


HIBEF26 66 871533 299716 610 1-508


HIBEF26 66 871533 299716 611 1-500


1098-1445


1472-2706


HOHBY04 71 888190 AL133245 612 1-427


479-789


120


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WO 01/55163 PCT/USO1/01358
1587-1740


3435-3548


6909-6992


8141-8476


9693-10005


12548-


13021


HOHBY04 71 888190 AL133245 613 1-516


HMKCH92 80 910936 AC026206 614 1-52


416-535


1184-1340


1502-1651


4581-4649


10517-


10753


10870-


10987


13190-


13220


HMKCH92 80 910936 AC034192 615 1-52


416-535


1184-1338


1500-1651


4583-4659


10526-


10754


10871-


10957


HMKCH92 80 910936 AC022381 616 1-52


416-535


1184-1340


1502-1651


4583-4651


10520-


10756


10873-


10990


13187-


13217


HMKCH92 80 910936 AC026206 617 1-107


HMKCH92 80 910936 AC022381 618 1-107


HWADD57 94 943039 AC011492 619 1-303


949-1648


1913-2937


3032-3231


3325-3443


4093-4485


4777-4936


5057-5548


121


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WO 01/55163 PCT/USO1/01358
5650-5968


HWADD57 94 943039 AC011492 620 1-50


852-907


988-1407


1584-1839


2455-2586


2689-2787


HBGMZ39 96 947112 AC008537 621 1-1186


HBGMZ39 96 947112 AC019337 622 1-1182


HBGMZ39 96 947112 AC008537 623 1-1993


2105-2385


2736-3068


4364-4489


6546-6781


7025-8165


HBGMZ39 96 947112 AC019337 624 1-1991


2103-2383


2734-3066


4360-4485


6541-6776


7021-8159


HBGMZ39 96 947112 AC008537 625 1-734


767-1001


HBGMZ39 96 947112 AC019337 626 1-158


291-565


598-832


HNTND64 104 954871 AC025090 627 1-465


HNTND64 104 954871 AC025090 628 1-454


HHAWC08 106 957942 AL096870 629 1-916


1032-1409


1680-1766


1866-2475


3074-3393


3538-3718


4038-4438


4643-4726


4816-4925


4972-6815


HHAWC08 106 957942 AL096870 630 1-1239


1381-1480


1568-1679


1853-1983


2020-2429


2491-3011


HHAWC08 106 957942 AL096870 631 1-283


975-1075


1551-1641


1837-1953


3100-3362


122


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WO 01/55163 PCT/USO1/01358
3825-3903


HMTAV95 117 614936 AL137000 632 1-47


321-642


2386-2508


3316-3493


4104-4572


6015-6340


6520-6573


HMTAV95 117 614936 AL137000 633 1-539


HMTAV95 117 614936 AL137000 634 1-510


HE9RA75 122 766779 AC009648 635 1-422


1494-15
84


HE9RA75 122 766779 AP002502 636 1-111


1088-1198


2267-2693


HE9RA75 122 766779 AP000788 637 1-111


1088-1198


2267-2693


HE9RA75 122 766779 AP000907 638 1-111


1088-1198


2267-2693


HE9RA75 122 766779 AC009648 639 1-2026


HE9R.A75 122 766779 AP002502 640 1-2336


HE9RA75 122 766779 AP000788 641 1-2299


HE9RA75 122 766779 AP000907 642 1-2346


HHFGP83 126 828162 AC026348 643 1-62


528-1080


1104-1284


1836-1940


2308-2633


HHFGP83 126 828162 AC026329 644 1-102


461-611


3520-3678


4094-4203


4670-5222


5246-5426


5978-6082


6450-6775


8830-9053


9755-10066


10098-


10279


10355-


10988


12072-


13795


HCQCI06 146 915000 AC068763 645 1-590


819-1083


HCQCI06 146 915000 AC069223 646 1-362


123


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WO 01/55163 PCT/USO1/01358
HCQCI06 146 915000 AC068763 647 1-593


HNFD052 149 916260 AC012307 648 1-1998


HWMEV63 157 931154 AC078816 649 1-1574


HEOST94 164 954582 AC021705 650 1-29


3 60-495


955-1079


1492-1559


1793-1954


2109-2237


8156-8313


9088-9598


9667-11499


12012-


12782


13030-


13154


13230-


13338


13623-


13713


13851-


13961


14179-


14392


14455-


14717


14747-


14846


15926-


17967


HEOST94 164 954582 AC006435 651 1-146


176-327


434-752


857-2289


2841-3106


3207-3592


3857-3983


4462-4550


4689-4800


5071-5244


5307-5574


5604-5703


6803-6986


7207-8866


HEOST94 164 954582 AC021705 652 1-304


HE8UT58 177 973153 AC032004 653 1-120


124


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WO 01/55163 PCT/USO1/01358
[46] Table 1B summarizes additional polynucleotides encompassed by the
invention
eluding cDNA clones related to the sequences (Clone m NO:Z), contig sequences
(contig
identifier (Contig >D:) contig nucleotide sequence identifiers (SEQ >D NO:X)),
and genomic
sequences (SEQ m NO:B). The first column provides a unique clone identifier,
"Clone >Z7
NO:Z", for a cDNA clone related to each contig sequence. The second column
provides the
sequence identifier, "SEQ >D NO:X", for each contig sequence. The third column
provides a
unique contig identifier, "Contig >D:" for each contig sequence. The fourth
column, provides
a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the
corresponding row of
the table. The fifth column provides the nucleotide sequence identifier, "SEQ
~ NO:B" for
a fragment of the BAC clone identified in column four of the corresponding row
of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide
position numbers)
within the polynucleotide sequence of SEQ m NO:B which delineate certain
polynucleotides
of the invention that are also exemplary members of polynucleotide sequences
that encode
polypeptides of the invention (e.g., polypeptides containing amino acid
sequences encoded
by the polynucleotide sequences delineated in column six, and fragments and
variants
thereof).
125


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
N G1 cn ~ ~D .-. t~ O t~ ~O ~ N ~
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CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
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CA 02395398 2002-06-20
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CA 02395398 2002-06-20
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CA 02395398 2002-06-20
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CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
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161


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
[47] Table 2 further characterizes certain encoded polypeptides of the
invention, by
providing the results of comparisons to protein and protein family databases.
The first
column provides a unique clone identifier, "Clone ID NO:", corresponding to a
cDNA clone
disclosed in Table 1A. The second column provides the unique contig
identifier, "Contig
ID:" which allows correlation with the information in Table 1A. The third
column provides
the sequence identifier, "SEQ ID NO:", for the contig polynucleotide
sequences. The fourth
column provides the analysis method by which the homology/identity disclosed
in the Table
was determined. The fifth column provides a description of the PFAM/NR hit
identified by
each analysis. Column six provides the accession number of the PFAM/NR hit
disclosed in
the fifth column. Column seven, score/percent identity, provides a quality
score or the
percent identity, of the hit disclosed in column five. Comparisons were made
between
polypeptides encoded by polynucleotides of the invention and a non-redundant
protein
database (herein referred to as "NR"), or a database of protein families
(herein referred to as
"PFAM"), as described below.
[48] The NR database, which comprises the NBRF PIR database, the NCBI GenPept
database, and the SIB SwissProt and TrEMBL databases, was made non-redundant
using the
computer program nrdb2 (Warren Gish, Washington University in Saint Louis).
Each of the
polynucleotides shown in Table 1A, column 3 (e.g., SEQ 117 NO:X or the 'Query'
sequence)
was used to search against the NR database. The computer program BLASTX was
used to
compare a 6-frame translation of the Query sequence to the NR database (for
information
about the BLASTX algorithm please see Altshul et al., J. Mol. Biol. 215:403-
410 (1990);
and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the
sequence that is most
similar to the Query sequence (the highest scoring 'Subject') is shown in
column five of
Table 2 and the database accession number for that sequence is provided in
column six. The
highest scoring 'Subject' is reported in Table 2 if (a) the estimated
probability that the match
occurred by chance alone is less than 1.0e-07, and (b) the match was not to a
known
repetitive element. BLASTX returns alignments of short polypeptide segments of
the Query
and Subject sequences which share a high degree of similarity; these segments
are known as
High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity
between the
Query and the Subject for each HSP as a percent identity in Column 7. The
percent identity is
determined by dividing the number of exact matches between the two aligned
sequences in
the HSP, dividing by the number of Query amino acids in the HSP and
multiplying by 100.
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The polynucleotides of SEQ >D NO:X which encode the polypeptide sequence that
generates
~n HSP are delineated by columns 8 and 9 of Table 2.
[49] The PFAM database, PFAM version 2.1, (Sonnhammer et al., Nucl. Acids
Res.,
26:320-322, 1998)) consists of a series of multiple sequence alignments; one
alignment for
each protein family. Each multiple sequence alignment is converted into a
probability model
called a Hidden Markov Model, or HMM, that represents the position-specific
variation
among the sequences that make up the multiple sequence alignment (see, e.g.,
Durbin et al.,
Biological sequence analysis: probabilistic models of proteins and nucleic
acids, Cambridge
University Press, 1998 for the theory of HMMs). The program HMMER version 1.8
(Sean
Eddy, Washington University in Saint Louis) was used to compare the predicted
protein
sequence for each Query sequence (SEQ ID NO:Y in Table 1A) to each of the HMMs
derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to
be a
significant match to a polypeptide of the invention if the score returned by
HMMER 1.8 was
greater than 0.8 times the HMMER 1.8 score obtained with the most distantly
related known
member of that protein family. The description of the PFAM family which shares
. a
significant match with a polypeptide of the invention is listed in column 5 of
Table 2, and
the database accession number of the PFAM hit is provided in column 6. Column
7 provides
the score returned by HMMER version 1.8 for the alignment. Columns 8 and 9
delineate the
polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which
show a
significant match to a PFAM protein family.
[50] As mentioned, columns 8 and 9 in Table 2, "NT From" and "NT To",
delineate the
polynucleotides of "SEQ >D NO:X" that encode a polypeptide having a
significant match to
the PFAM/NR database as disclosed in the fifth column. In one embodiment, the
invention
provides a protein comprising, or alternatively consisting of, a polypeptide
encoded by the
polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2. Also
provided
are polynucleotides encoding such proteins, and the complementary strand
thereto.
[51] The nucleotide sequence SEQ >D NO:X and the translated SEQ ID NO:Y are
sufficiently accurate and otherwise suitable for a variety of uses well known
in the art and
described further below. For instance, the nucleotide sequences of SEQ >D NO:X
are useful
for designing nucleic acid hybridization probes that will detect nucleic acid
sequences
contained in SEQ >D NO:X or the cDNA contained in Clone >D NO:Z. These probes
will
also hybridize to nucleic acid molecules in biological samples, thereby
enabling immediate
applications in chromosome mapping, linkage analysis, tissue identification
and/or typing,
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CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
and a variety of forensic and diagnostic methods of the invention. Similarly,
polypeptides
Identified from SEQ ID NO:Y may be used to generate antibodies which bind
specifically to
these polypeptides, or fragments thereof, and/or to the polypeptides encoded
by the cDNA
clones identified in, for example, Table 1A.
[52] Nevertheless, DNA sequences generated by sequencing reactions can contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or deletions
of nucleotides in the generated DNA sequence. The erroneously inserted or
deleted
nucleotides cause frame shifts in the reading frames of the predicted amino
acid sequence. In
these cases, the predicted amino acid sequence diverges from the actual amino
acid sequence,
even though the generated DNA sequence may be greater than 99.9% identical to
the actual
DNA sequence (for example, one base insertion or deletion in an open reading
frame of over
1000 bases).
[53] Accordingly, for those applications requiring precision in the nucleotide
sequence
or the amino acid sequence, the present invention provides not only the
generated nucleotide
sequence identified as SEQ ID NO:X, and a predicted translated amino acid
sequence
identified as SEQ m NO:Y, but also a sample of plasmid DNA containing cDNA
Clone >D
NO:Z (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation
numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and
having
depositor reference numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth,
for example,
in Table 1 A, 6 and 7). The nucleotide sequence of each deposited clone can
readily be
determined by sequencing the deposited clone in accordance with known methods.
Further,
techniques known in the art can be used to verify the nucleotide sequences of
SEQ >D NO:X.
[54] The predicted amino acid sequence can then be verified from such
deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone
can also be
directly determined by peptide sequencing or by expressing the protein in a
suitable host cell
containing the deposited human cDNA, collecting the protein, and determining
its sequence.
RACE Protocol For Recovery of Full-Length Genes
[55] Partial cDNA clones can be made full-length by utilizing the rapid
amplification of
cDNA ends (RACE) procedure described in Frohman, M.A., et al., Proc. Nat'l.
Acad. Sci.
USA, 85:8998-9002 ( 1988). A cDNA clone missing either the 5' or 3' end can be
reconstructed to include the absent base pairs extending to the translational
start or stop
codon, respectively. In some cases, cDNAs are missing the start codon of
translation,
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CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
therefor. The following briefly describes a modification of this original 5'
RACE procedure.
Poly A+ or total RNA is reverse transcribed with Superscript II (GibcoBRL) and
an
antisense or complementary primer specific to the cDNA sequence. The primer is
removed
from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA
is then
tailed with dATP and terminal deoxynucleotide transferase (GibcoBRL). Thus, an
anchor
sequence is produced which is needed for PCR amplification. The second strand
is
synthesized from the dA-tail in PCR buffer, Taq DNA polymerise (Perkin-Elmer
Cetus), an
oligo-dT primer containing three adjacent restriction sites (XhoI, SaII and
CIaI) at the 5' end
and a primer containing just these restriction sites. This double-stranded
cDNA is PCR
amplified for 40 cycles with the same primers as well as a nested cDNA-
specific antisense
primer. The PCR products are size-separated on an ethidium bromide-agarose gel
and the
region of gel containing cDNA products the predicted size of missing protein-
coding DNA is
removed. cDNA is purified from the agarose with the Magic PCR Prep kit
(Promega),
restriction digested with XhoI or SaII, and ligated to a plasmid such as
pBluescript SKII
(Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria
and the
plasmid clones sequenced to identify the correct protein-coding inserts.
Correct 5' ends are
confirmed by comparing this sequence with the putatively identified homologue
and overlap
with the partial cDNA clone. Similar methods known in the art and/or
commercial kits are
used to amplify and recover 3' ends.
[56] Several quality-controlled kits are commercially available for purchase.
Similar
reagents and methods to those above are supplied in kit form from GibcoBRL for
both 5' and
3' RACE for recovery of full length genes. A second kit is available from
Clontech which is
a modification of a related technique, SLIC (single-stranded ligation to
single-stranded
cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The
major
differences in procedure are that the RNA is alkaline hydrolyzed after reverse
transcription
and RNA ligase is used to join a restriction site-containing anchor primer to
the first-strand
cDNA. This obviates the necessity for the dA-tailing reaction which results in
a polyT
stretch that is difficult to sequence past.
[57] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library
double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is
synthesized
with an antisense cDNA-specific primer and a plasmid-anchored primer. These
primers are
removed and a symmetric PCR reaction is performed with a nested cDNA-specific
antisense
primer and the plasmid-anchored primer.
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CA 02395398 2002-06-20
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itNA Ligase Protocol For Generating The 5' or 3' Erzd Sequences To Obtain
Frrll Ler:gtJr
Genes
[58J Once ,a gene of interest is identified, several methods are available for
the
identification of the S' or 3' portions of the gene which may not be present
in the original
cDNA plasmid. These methods include, but are not limited to, filter probing,
clone
enrichment using specific probes and 'protocols similar and identical to 5'
and 3' RACE.
While the full length gene may be present in the library and can be identified
by probing, a
useful method for generating the 5' or 3' end is to use the existing sequence
information from
the original cDNA to generate the missing information. A method similar to 5'
RACE is
available for generating the missing 5' end of a desired full-length gene.
(This method was
published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684
(1993)). Briefly, a
specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA
presumably
containing full-length gene RNA transcript and a primer set containing a
primer specific to
the ligated RNA oligonucleotide and a primer specific to a known sequence of
the gene of
interest, is used to PCR amplify the S' portion of the desired full length
gene which may then
be sequenced and used to generate the full length gene. This method starts
with total RNA
isolated from the desired source, poly A RNA may be used but is not a
prerequisite for this
procedure. The RNA preparation may then be treated with phosphatase if
necessary to
eliminate 5' phosphate groups on degraded or damaged RNA which may interfere
with the
later RNA ligase step. The phosphatase if used is then inactivated and the RNA
is treated
with tobacco acid pyrophosphatase in order to remove the cap structure present
at the 5' ends
of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of
the cap
cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA
ligase.
This modified RNA preparation can then be used as a template for first strand
cDNA
synthesis using a gene specific oligonucleotide. The first strand synthesis
reaction can then
be used as a template for PCR amplification of the desired 5' end using a
primer specific to
the ligated RNA oligonucleotide and a primer specific to the known sequence of
the gene of
interest. The resultant product is then sequenced and analyzed to confirm that
the 5' end
sequence belongs to the relevant gene.
[59] The present invention also relates to vectors or plasmids which include
such DNA
sequences, as well as the use of the DNA sequences. The material deposited
with the ATCC
(deposited with the ATCC on October 5, 2000, and receiving ATCC designation
numbers
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PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and
receiving
ATCC designation numbers TS-l, TS-2, AC-1, and AC-2; and/or as set forth, for
example, in
Table 1A, Table 6, or Table 7) is a mixture of cDNA clones derived from a
variety of human
tissue and cloned in either a plasmid vector or a phage vector, as described,
for example, in
Table 7. These deposits are referred to as "the deposits" herein. The tissues
from which
some of the clones were derived are listed in Table 7, and the vector in which
the
corresponding cDNA is contained is also indicated in Table 7. The deposited
material
includes cDNA clones corresponding to SEQ >D NO:X described, for example, in
Table 1 A
(Clone >D NO:Z). A clone which is isolatable from the ATCC Deposits by use of
a sequence
listed as~ SEQ ID NO:X, may include the entire coding region of a human gene
or in other
cases such , clone may include a substantial portion of the coding region of a
human gene.
Furthermore, although the sequence listing may in some instances list only a
portion of the
DNA sequence in a clone included in the ATCC Deposits, it is well within the
ability of one
skilled in the art to sequence the DNA included in a clone contained in the
ATCC Deposits
by use of a sequence (or portion thereof) described in, for example Tables
lAor 2 by
procedures hereinafter further described, and others apparent to those skilled
in the art.
[60] Also provided in Table 7 is the name of the vector which contains the
cDNA
clone. Each vector is routinely used in the art. The following additional
information is
provided for convenience.
[61] Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and
5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-
7600 (1988);
Along-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK
(Alting-
Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from
Stratagene
Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037. pBS
contains an
ampicillin resistance gene and pBK contains a neomycin resistance gene.
Phagemid pBS
may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK
may be
excised from the Zap Express vector. Both phagemids may be transformed into E.
coli strain
XL-1 Blue, also available from Stratagene.
[62] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were
obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897.
All Sport
vectors contain an ampicillin resistance gene and may be transformed into E.
coli strain
DHIOB, also available from Life Technologies. See, for instance, Gruber, C.
E., et al., Focus
167


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
15:59- (1993). Vector lafmid BA (Bento Soares, Columbia University, New York,
NY)
contains an ampicillin resistance gene and can be transformed into E. coli
strain XL-1 Blue.
Vector pCR~2.1, which is available from Invitrogen, 1600 Faraday Avenue,
Carlsbad, CA
92008, contains an ampicillin resistance gene and may be transformed into E.
coli strain
DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc.
Acids Res.
16:9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (1991).
[63] The present invention also relates to the genes corresponding to SEQ ID
NO:X,
SEQ ID NO:Y, and/or the deposited clone (Clone ID NO:Z). The corresponding
gene can be
isolated in accordance with known methods using the sequence information
disclosed herein.
Such methods include preparing probes or primers from the disclosed sequence
and
identifying or amplifying the corresponding gene from appropriate sources of
genomic
material.
[64] Also provided in the present invention are allelic variants, orthologs,
and/or
species homologs. Procedures known in the art can be used to obtain full-
length genes, allelic
variants, splice variants, full-length coding portions, orthologs, and/or
species homologs of
genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides
encoded by
genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA
contained in Clone ID NO:Z, using information from the sequences disclosed
herein or the
clones deposited with the ATCC. For example, allelic variants and/or species
homologs may
be isolated and identified by making suitable probes or primers from the
sequences provided
herein and screening a suitable nucleic acid source for allelic variants
and/or the desired
homologue.
[65] The polypeptides of the invention can be prepared in any suitable manner.
Such
polypeptides include isolated naturally occurring polypeptides, recombinantly
produced
polypeptides, synthetically produced polypeptides, or polypeptides produced by
a
combination of these methods. Means for preparing such polypeptides are well
understood in
the art.
[66] The polypeptides may be in the form of the secreted protein, including
the mature
form, or may be a part of a larger protein, such as a fusion protein (see
below). It is often
advantageous to include an additional amino acid sequence which contains
secretory or
leader sequences, pro-sequences, sequences which aid in purification, such as
multiple
histidine residues, or an additional sequence for stability during recombinant
production.
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CA 02395398 2002-06-20
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[67] The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purified. A recombinantly produced
version of a
polypeptide, including the secreted polypeptide, can be substantially purified
using
techniques described herein or otherwise known in the art, such as, for
example, by the one-
step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides
of the
invention also can be purified from natural, synthetic or recombinant sources
using
techniques described herein or otherwise known in the art, such as, for
example, antibodies of
the invention raised against the polypeptides of the present invention in
methods which are
well known in the art.
[68] ' The present invention provides a polynucleotide comprising, or
alternatively
consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence
contained in Clone >D NO:Z. The present invention also provides a polypeptide
comprising,
or alternatively, consisting of, the polypeptide sequence of SEQ >D NO:Y, a
polypeptide
encoded by SEQ 1~ NO:X or a complement thereof, a polypeptide encoded by the
cDNA
contained in Clone >D NO:Z, and/or the polypeptide sequence encoded by a
nucleotide
sequence in SEQ >D NO:B as defined in column 6 of Table 1B. Polynucleotides
encoding a
polypeptide comprising, or alternatively consisting of the polypeptide
sequence of SEQ ID
NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA
contained in Clone >D NO:Z, and/or a polypeptide sequence encoded by a
nucleotide
sequence in SEQ 1D NO:B as defined in column 6 of Table 1B are also
encompassed by the
invention. The present invention further encompasses a polynucleotide
comprising, or
alternatively consisting of, the complement of the nucleic acid sequence of
SEQ ID NO:X, a
nucleic acid sequence encoding a polypeptide encoded by the complement of the
nucleic acid
sequence of SEQ m NO:X, and/or the cDNA contained in Clone m NO:Z.
[69] Moreover, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of
the sequences delineated in Table 1B column 6, or any combination thereof.
Additional,
representative examples of polynucleotides of the invention comprise, or
alternatively consist
of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the
complementary
strands) of the sequences delineated in Table 1B column 6, or any combination
thereof. In
further embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated in Table 1B, column 6, and have
a nucleic acid
sequence which is different from that of the BAC fragment having the sequence
disclosed in
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SEQ >D NO:B (see Table 1B, column 5). In additional embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
Table 1B, column 6, and have a nucleic acid sequence which is different from
that published
for the BAC clone identified as BAC m NO:A (see Table 1B, column 4). In
additional
embodiments, the above-described polynucleotides of ~ the invention comprise,
or
alternatively consist of, sequences delineated in Table 1 B, column 6, and
have a nucleic acid
sequence which is different from that contained in the BAC clone identified as
BAC m
NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
are also 'encompassed by the invention. Additionally, fragments and variants
of the above-
described polynucleotides and polypeptides are also encompassed by the
invention.
[70] Further, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
Clone 1D NO:Z
(see Table 1 B, column 1 ), or any combination thereof. Additional,
representative examples
of polynucleotides of the invention comprise, or alternatively consist of,
one, two, three, four,
five, six, seven, eight, nine, ten, or more of the complementary strands) of
the sequences
delineated in column 6 of Table 1B which correspond to the same Clone >D NO:Z
(see Table
1B, column 1), or any combination thereof. In further embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
column 6 of Table 1B which correspond to the same Clone >D NO:Z (see Table 1B,
column
1) and have a nucleic acid sequence which is different from that ofthe BAC
fragment having
the sequence disclosed in SEQ m NO:B (see Table 1B, column S). In additional
embodiments, the above-described polynucleotides of the invention comprise, or
alternatively consist of, sequences delineated in column 6 of Table 1B which
correspond to
the same Clone >D NO:Z (see Table 1B, column 1) and have a nucleic acid
sequence which is
different from that published for the BAC clone identified as BAC >D NO:A (see
Table 1B,
column 4). In additional embodiments, the above-described polynucleotides of
the invention
comprise, or alternatively consist of, sequences delineated in column 6 of
Table 1 B which
correspond to the same Clone >D NO:Z (see Table 1B, column 1) and have a
nucleic acid
sequence which is different from that contained in the BAC clone identified as
BAC m
NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
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are also encompassed by the invention. Additionally, fragments and variants of
the above-
'described polynucleotides and polypeptides are also encompassed by the
invention.
[71] Further, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
contig sequence
identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof.
Additional,
representative examples of polynucleotides of the invention comprise, or
alternatively consist
of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the
complementary
strands) of the sequences delineated in column 6 of Table 1B which correspond
to the same
contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any
combination
thereof. In further embodiments, the above-described polynucleotides of the
invention
comprise, or alternatively consist of, sequences delineated in column 6 of
Table 1B which
correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B,
column 2)
and have a nucleic acid sequence which is different from that of the BAC
fragment having
the sequence disclosed in SEQ ID NO:B (see Table 1B, column S). In additional
embodiments, the above-described polynucleotides of the invention comprise, ,
or
alternatively consist of, sequences delineated in column 6 of Table 1B which
correspond to
the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2) and
have a
nucleic acid sequence which is different from that published for the BAC clone
identified as
BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
column 6 of Table 1B which correspond to the same contig sequence identifer
SEQ ID NO:X
(see Table 1 B, column 2) and have a nucleic acid sequence which is different
from that
contained in the BAC clone identified as BAC ID NO:A (See Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention. Additionally, fragments and variants of the above-described
polynucleotides and
polypeptides are also encompassed by the invention.
[72] Moreover, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of
the sequences delineated in the same row of Table 1B column 6, or any
combination thereof.
Additional, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
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complementary strands) of the sequences delineated in the same row of Table 1B
column 6,
6r any combination thereof. In preferred embodiments, the polynucleotides of
the invention
comprise, or alternatively consist of, one, two, three, four, five, six,
seven, eight, nine, ten, or
more of the complementary strands) of the sequences delineated in the same row
of Table
1B column 6, wherein sequentially delineated sequences iri the table (i.e.
corresponding to
those exons located closest to each other) are directly contiguous in a 5' to
3' orientation. W
further embodiments, above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated in the same row of Table 1B,
column 6, and
have a nucleic acid sequence which is different from that of the BAC fragment
having the
sequence disclosed in SEQ >D NO:B (see Table 1B, column 5). In additional
embodiments,
the above-described polynucleotides of the invention comprise, or
alternatively consist of,
sequences delineated in the same row of Table 1B, column 6, and have a nucleic
acid
sequence which is different from that published for the BAC clone identified
as BAC >I7
NO:A (see Table 1B, column 4). In additional embodiments, the above-described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
the same row of Table 1B, column 6, and have a nucleic acid sequence which is
different
from that contained in the BAC clone identified as BAC >D NO:A (see Table 1B,
column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention.
[73] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B, and the polynucleotide sequence
of SEQ m
NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants
thereof. Polypeptides
encoded by these polynucleotides, other polynucleotides that encode these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
[74] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
Clone ID NO:Z
(see Table 1B, column 1), and the polynucleotide sequence of SEQ B7 NO:X
(e.g., as defined
in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments,
the
delineated sequences) and polynucleotide sequence of SEQ >Z7 NO:X correspond
to the
same Clone ID NO:Z. Polypeptides encoded by these polynucleotides, other
polynucleotides
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that encode these polypeptides, and antibodies that bind these polypeptides
are also
encompassed by the invention.
[75] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in the same row of column 6 of Table 1B, and the
polynucleotide
sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or
variants
thereof. In preferred embodiments, the delineated sequences) and
polynucleotide sequence
of SEQ ID NO:X correspond to the same row of column 6 of Table 1B.
Polypeptides
encoded by these polynucleotides, other polynucleotides that encode these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
[76] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of the
sequence of SEQ >D NO:X are directly contiguous. Nucleic acids which hybridize
to the
complement of these 20 contiguous polynucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids that encode these polypeptides, and antibodies that bind
these
polypeptides are also encompassed by the invention. Additionally, fragments
and variants of
the above-described polynucleotides, nucleic acids, and polypeptides are also
encompassed
by the invention.
(77] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of a
fragment or variant of the sequence of SEQ >D NO:X are directly contiguous
Nucleic acids
which hybridize to the complement of these 20 contiguous polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention. Polypeptides encoded by these polynucleotides
and/or
nucleic acids, other polynucleotides and/or nucleic acids encoding these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
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[78] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of the
sequence of SEQ ~ NO:X and the 5' 10 polynucleotides of the sequence of one of
the
sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic
acids which
hybridize to the complement of these 20 contiguous polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention. Polypeptides encoded by these polynucleotides
and/or
nucleic acids, other polynucleotides and/or nucleic acids encoding these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
[79] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of a
fragment or variant of the sequence of SEQ >D NO:X and the 5' 10
polynucleotides of the
sequence of one of the sequences delineated in column 6 of Table 1B are
directly contiguous.
Nucleic acids which hybridize to the complement of these 20 contiguous
polynucleotides
under stringent hybridization conditions or alternatively, under lower
stringency conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides
and/or nucleic acids, other polynucleotides and/or nucleic acids encoding
these polypeptides,
and antibodies that bind these polypeptides are also encompassed by the
invention.
Additionally, fragments and variants of the above-described polynucleotides,
nucleic acids,
and polypeptides, are also encompassed by the invention.
[80] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1 B and the 5' 10
polynucleotides of another
sequence in column 6 are directly contiguous. Nucleic acids which hybridize to
the
complement of these 20 contiguous polynucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids encoding these polypeptides, and antibodies that bind
these polypeptides
are also encompassed by the invention. Additionally, fragments and variants of
the above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by the
W venrion.
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[81] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another
sequence in column 6 corresponding to the same Clone ID NO:Z (see Table 1B,
column 1)
are directly contiguous. Nucleic acids which hybridize to the complement of
these 20 lower
stringency conditions, are also encompassed by the invention. Polypeptides
encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids encoding
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention. Additionally, fragments and variants of the above-described
polynucleotides,
nucleic acids, and polypeptides are also encompassed by the invention.
[82] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one
sequence in column 6 corresponding to the same contig sequence identifer SEQ
ID NO:X
(see Table 1B, column 2) are directly contiguous. Nucleic acids which
hybridize to the
complement of these 20 contiguous polynucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids encoding these polypeptides, and antibodies that bind
these polypeptides
are also encompassed by the invention. Additionally, fragments and variants of
the above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by the
invention.
[83] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another
sequence in column 6 corresponding to the same row are directly contiguous. In
preferred
embodiments, the 3' 10 polynucleotides of one of the sequences delineated in
column 6 of
Table 1B is directly contiguous with the 5' 10 polynucleotides of the next
sequential exon
delineated in Table 1B, column 6. Nucleic acids which hybridize to the
complement of these
20 contiguous polynucleotides under stringent hybridization conditions or
alternatively,
under lower stringency conditions, are also encompassed by the invention.
Polypeptides
encoded by these polynucleotides and/or nucleic acids, other polynucleotides
and/or nucleic
acids encoding these polypeptides, and antibodies that bind these polypeptides
are also
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encompassed by the invention. Additionally, fragments and variants of the
above-described
polynucleotides, nucleic acids, and polypeptides are also encompassed by the
invention.
[84J Many polynucleotide sequences, such as EST sequences, are publicly
available
and accessible through sequence databases and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are specifically
excluded from the scope of the present invention. Accordingly, for each contig
sequence
(SEQ >D NO:X) listed in the fourth column of Table 1 A, preferably excluded
are one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of a-b,
where a is any integer between 1 and the final nucleotide minus 15 of SEQ >D
NO:X, b is an
integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b
correspond to the
positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater
than or equal
to a + 14. More specifically, preferably excluded are one or more
polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a and b are
integers as defined in columns 4 and 5, respectively, of Table 3. In specific
embodiments, the
polynucleotides of the invention do not consist of at least one, two, three,
four, five, ten, or
more of the specific polynucleotide sequences referenced by the Genbank
Accession No. as
disclosed in column 6 of Table 3 (including for example, published sequence in
connection
with a particular BAC clone). In further embodiments, preferably excluded from
the
invention are the specific polynucleotide sequences) contained in the clones
corresponding
to at least one, two, three, four, five, ten, or more of the available
material having the
accession numbers identified in the sixth column of this Table (including for
example, the
actual sequence contained in an identified BAC clone). In no way is this
listing meant to
encompass all of the sequences which may be excluded by the general formula,
it is just a
representative example. All references available through these accessions are
hereby
incorporated by reference in their entirety.
TABLE 3
SE


Q


ID


Clone ID NO: Contig EST Disclaimer


NO: Z X ID: Range ACCesslon #'s
of a
Range
of b


HAJAU21 11 1007572 1 - 628 1 S - AL045231, AI953200,
642


AW005541, AA513506,


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220296, Y 12226,


AB015317, X54424,


AJ224112, AJ224114,


A74844, and AJ224113.


HMSKF13 12 708207 1 - 387 15 - 401 AA148393, AC023891,
and


AC023891.


HHPFV44 13 1121866 1 - 683 15 - 697 898212, F06274, 887869,


and AF225971.


HLHCT68 14 764745 1 - 425 15 - 439 891150, H67895, AI205078,


225012, C05212, 230134,


AA194359, AC010344,


AC010344, AC010344,


AC008496, and AC008496.


HTLAQ18 15 811792 1 - 649 15 - 663 AA442624, AI382107,


AA009701, and AL133054.


HE6FD03 16 1153880 1 - 837 15 - 851 AA431537, AW404444,


AI283081, AI355127,


AA431213, AI241247,


AI025060, AI017479,


AI276732, AA737782,


AI351906, AA700252,


AI564874, AA861742,


AI864024, AI051964,


240548, AA976182,


AI363484, AI350761,


AA688143, AI160881,


AA662752, AI028241,


884964, AI332354, F08983,


AI471267, 878868,


AA670442, AI920956,


AA312704, AC000097,


AC006547, AC000083,


AC003060, AC005664,
and


AJ236691.


HEQAY32 17 869178 1 - 1169 15 - 1183AA459604, AA703415,


AA703508, AI337830,


AI924185, AA993540,


AA398958, AA047107,


AI581879, 850486,


AA399541, AA826082,


AA047244, AI570681,


AW007699, AA742413,


AI278602, AI479681,


AA810070, AI346375,


AI468327, AA766769,


AI721136, AA317334,


AI378094, H08229,


AA878088, AI269575,


T36249, AA878089,


177


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M78847, H08868,


AW 138078, AA322162,


AI824732, F04540,
F04098,


AI383526, F02816,
850380,


AA383971, AI444981,


AI953863, and AB033004.


HSSJM44 18 871067 1 - 1034 15 - 1048 AA402834, AA313382,
~


T75200, H10173, 246198,


T34442, 813072,


AA357096, AA383450,


T07941, AC026352,


AC026352, AC069443,


AC069443, AC068755,
and


AC068755.


HOFMT55 19 1083824 1 - 663 15 - 677


HYAAU65 20 909956 1 - 581 15 - 595 T81523, T98761,


AL080117, AB023176,


U14103Nov 22 2000


3:26:56: and 380AM.


HAHEF22 21 910996 1 - 872 15 - 886 AA447298.


HPDV067 22 1173157 1 - 1135 15 - 1149 AI963225, AI758877,


AI807994, AW 150122,


AI248930, AA292182,


AI708051, AI142918,


AA993485, AW001319,


AI798505, AI218504,


AA983431, AA196524,


AA633225, AI306679,


848227, AA897389,


AA399226, AI734849,


854747, AW072089,


AA405765, AA476847,


AA405846, 848226,


AA865971, AA402159,


AA290822, AA954879,


AA291888, AW082573,


AA410677, AA402040,


AA235386, AA954094,


AA284936, AI471266,


AW374525, AW374552,


AW351832, AI267732,


AW374499, AW374468,


AC005954, AF023617,
and


261317.


HFKKS58 23 1152246 1 - 1444 15 - 1458 AW246359; AW246572,


AA827562, AA514488,


AL135673, AI539185,


AA459956, AI190270,


AA778031, AA083889,


178


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AI539830, AL134250,


AA255533, AA460045,


AA532881, AA083888,


F19104, AA384265,


AA749416, AI972095,


AA247961, AA702934,


N66268, AA364111,


AI630888, AA255505,


AW438881, N79931,


AA729375, AA334602,


AW363733, T06791,
and


AF116910.


HTFBE02 24 1199605 1 - 1364 15 - 1378 AI923217, AA197141,


AI814569, AI762903,


AI804682, AI804663,


AI143304, AA197116,


AI082030, AI911904,


AI139520, 892413,


AW274978, W93272,


AI183410, AW450838,


AI216343, AA680119,


AI918971, C15320,


W93273, F00607,


AI823928, T51116,
219397,


T96093, 892414, T51024,


D53214, AI422647,


AI216344, AI741425,


AI936703, T96094,
842713,


AI869077, F00114,
C00216,


817362, 266165, and


256024.


HCEOR02 25 921110 1 - 545 15 - 559 AA325666.


HLMD095 26 928344 1 - 469 15 - 483 AC020641.


HCEMY90 27 932927 1 - 592 15 - 606 AA324130, AA361570,


812848, AF214633,


AF214633, AC024242,
and


AC024242.


HBGQN46 28 945370 1 - 888 15 - 902 AA479990, AW293680,


AI473367, AI184880,


AI086741, AI081183,


AI214276, AA742259,


AA836754, 855060,


AA251267, AI285889,


AI813391, AW297831,


AI819671, AF038458,
and


AF038458.


HTEPK69 29 951188 1 - 1255 15 - 1269 AL046787, 278359,


824919, H62992, W39450,


H24270, AA364532,


179


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. 241904, AA305651,
AI739068, AA325114,
AI750479, AA101388,
AA346082, AI469164,
AA417722, AA076212,
AI016444, AB029031,
Y17923, and I86429.


HRODF07 30 952426 1 - 324 15 - 338 ACOI 1597, and AC023402.


HUJDC24 31 953517 1 - 1408 15 - 1422 AI467849, AI742229,


AW148851, AI916736,


AI281433, AA804534,


AI564217, AI523942,


AW364769, AW299493,


AI620008, AI123945,


AI377579, 278359,


AI191913, AI610109,


AW085483, AA923067,


AI769651, AI928062,


AI078678, AA854786,


AI025849, AI581271,


AA609195, AA812157,


AI150639, AI261346,


AI433379, AA599239,


AI743119, AW 194073,


AI760076, AA507048,


AI041747, AW002493,


AW391749, AI492025,


AA890051, AA113313,


AA004830, C06203,


AW 169615, AA814762,


C75084, AI814568,


AA631549, AW408517,


AA079540, AI245591,


AA317350, AI025449,


AI017117, H62870,


AL046787, AW269195,


AA953576, AA083323,


AW079439, AI655457,


AA890518, H22877,


AA861351, AA768236,


AA079508, H65006,


AW075096, H65005,


AI538604, T30796,


AA004983, AA384663,


AA380072, AA364532,


AA180878, AA054836,


867437, 238201,


AA648532, W38371,


AA180892, AW407493,


180


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AI739068, 824919, H62992,
r AB029031, Y17923, and

I86429.


HNHCI32 32 861673 1 - 586 15 - 600 AF112462, and AR035954.


HUVDR03 33 1212685 1 - 3158 15 - 3172AA768846, AI671768,


AI961239, AI884699,


AA514474, AI679628,


AI521280, AA156958,


AI139530, AI885479,


AI336225, AI186828,


AI735075, AI626081,


AI494518, AI275985,


AA749446, AI914010,


w AI921928, AW207720,


AI130746, AA993271,


AI130728, AI299980,


AA976621, W69667,


AI279330, AA229014,


AA742981, 819745,


AA156866, AI216523,


AA255782, AI811823,


884393, AA488821,


AI419263, AA489068,


AA229863, AA701250,


AA705638, N47318,


AA229731, AW271763,


AA687125, AA862901,


AL044304, AW372345,


AW272381, AA939081,


W73428, AI478491,


AW089880, AA702739,


AA765337, AW021871,


F06156, AA977247,


AI141957, W73367,


AI283582, W45513,


AA127066, 884392,


AI985183, AW439593,


AI560741, AI687916,


AI186368, AI203537,


AA047572, W69666,


H66133, AI081094,


AA453238, AA463404,


AW294792, AA356896,


AA318684, H66549,


AL046248, AA373158,


AA011074, AL046015,


225224, AA480226,


AW292636, H43300,


AI222506, W24708,


181


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA492470, T47922,


AA579885, T47923,


AA348753, T05906,


AA455850, H43299,


AW292633, AA011075,


AI419633, D61430,


AA772416, AI587496,


AA147480, N92391,


AA378964, 845164,


AA714016, AA130104,


AI560507, AW272813,


AW376847, AI471022,


H16172, AI568064,
868334,


AI567645, AW376809,


W45654, AA256004,


AA280280, AL133683,


AA378963, AA341472,


AA887264, AA147880,


AA704633, AA429202,


AI905673, AI216605,


AW168495, C14179,


AI963999, AC020663,


U70255, L06564, X89268,


and AF005380.


HLTCT21 34 975033 1 - 1305 15 - 1319 AA001257, AW390109,


AA149562, C05730,


AI342264, AI274957,


AI139524, AA151642,


AA467756, AI132915,


AW390186, AI683026,


AA467995, AI921624,


AA002262, AI624444,


AI302167, AI347041,


AI382066, AI888857,


AI041643, AI283206,


AI160682, AI393801,


AW391467, T93307,


AA379905, AI094800,


AA856948, AW391475,


AI312598, AI000325,


AI289177, AI310436,


H15978, AA565691,


AA885184, AI278759,


AI342194, AI083948,


AI921687, AI347545,


D83882, AA453766,


C05795, AW297678,


AA922168, N94588,


AI801674, AI679825,


182


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW008455, AA358043,


AI679249, AAl 15793,


AI697006, AI493351,


AI630853, W60406,


W52946, AI351551,


AA453850, AI302459,


AI907019, AW365995,


AW365918, AA862930,


AW381563, AW365896,


AW069527, AA467938,


AI421230, AA358042,


T30922, AW082407,


AI559167, AI784345,


AA889377, AI174263,


T08780, AI126061,


AA468056, AI094399,


AW379998, T93984,


AI264468, AI378984,


239023, AF010127,


A84918, AF015450,


A86556, AF009618,


AF041458, A84924,


AF005774, AF041461,


AF041460, AF015451,


AF041459, AF009616,


AF041462, AC007283,


AF009617, AF009619,


AF015452, U97075,


A84916, AF005775, and


A86558.


HLHGH34 35 1023772 1 - 1529 15 - 1543AI056280, AA399001,


N31314, AL120264,


AA399591, 895732,


AA224343, 858162,


AA699899, AJ223333,


AF045888, and AF012128.


HFXBN61 36 1172816 1 - 1255 15 - 1269AA478265, AA428837,


AA496285, AA478321,


817757, 837358, 867163,


AA400943, AA400877,


819123, 811880, 844789,


and U87305.


HSLFT94 37 1199828 1 - 1387 15 - 1401


HELGW31 38 610003 1 - 1645 15 - 1659C14389, D80268,


AW 177440, AW 177501,


AW177511, AW352117,


D81026, D59502, AI905856,


AW 178893, T03269,


C14014, AA305578,


183


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW179328, AW366296,


AW360811, AW375405,


r
AA514188, D58283,


D59859, D80022, C14331,


D80166, D80195, D80193,


D59927, D59467, D51423,


D59619, D80210, D51799,


D80391, D80164, D59275,


D80240, D80253, D80043,


D59787, D80227,


AW378532, D81030,


D80212, D80196, D80188,


D80219, AW176467,


w C15076, D80269, D80038,


D59610, D57483, D80366,


AA305409, C14429,


D51022, D50979, D50995,


D59889, AW 178762,


D80024, AW377671,


D80378, AW 178775,


AW360844, AW360817,


D80241, D51060,


AW352158, AW375406,


D80248, AW378534,


AW179332, AW377672,


AW 179023, AW 178905,


D80134, D80045, D80132,


D51097, AW352170,


D58253, AW352171,


D80522, AW377676,


AW 177731, AW 178907,


AW 179019, AW 179024,


D80251, AA514186,


C75259, D80133,


AW 178906, AW 177505,


AW 179020, AW 178909,


AW 177456, AW 179329,


AW 178980, AW 177733,


AW378528, AW 178908,


AW 178754, AW 179018,


AW 179004, AW 178914,


AW178911, AW367967,


AW352174, D80302,


AW 178774, AW 177723,


D80439, D80247, T48593,


AI535850, AW178983,


D51103, AW367950,


C 14975, AW 178986,


D45260, AI525913, Y17188,


184


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
X82626, A84916, A67220,


D89785, A62300, A62298,


A78862, D34614, D26022,


D88547, AJ132110,


AR018138, X67155,


A25909, AF058696,


AR008278, Y12724,


AR025207, AB028859,


AB012117, A94995,


A85396, D88507,


AR066482, A44171,


A85477, AR008443,
I19525,


A86792, I18367, X93549,


I50126, I50132, I50128,


I50133, AR066488,
A82595,


AR066490, AR016514,


D50010, D13509,


AR060138, A45456,


A26615, AR052274,


Y09669, AR060385,


AB002449, AR066487,


A43192, A43190,


AR038669, A30438,


AR008408, U79457,


AF135125, AR060133,


AR008382Nov 22 2000


12:53:15: and 800AM.


HDJME16 39 1206706 1 - 155915 - 1573 AL046450, AW296638,


AW027431, N25289,


AA653247, AA916161,


AA343865, AF135440,
and


AL137459.


HISAR63 40 1199655 1 - 162915 - 1643 AA778721, AI309326,


AI923088, AW 157189,


AW009559, N21676,


AW277241, AI015567,


AA142926, AA935517,


AI340068, N71214,


AA046446, AI890415,


AA313266, AI760248,


AI421490, AI423473,


AA576688, AI342399,


AA946956, AW 163161,


AA826534, AA143149,


AA056157, AI803454,


AA125818, AA233629,


N22011, AA594414,


AA405935, AI862039,


AA315935, AW148924,


185


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI245757, AW374039,


N80237, AA488928,


AW304989, W33046,


AA782164, AW403205,


H89121, T78059, AI953431,


AI344475, AA074821,


AI148049, AI707964,


AA864308, AA702036,


AA058701, AA034997,


AW057651, AA045662,


AA084570, AA172038,


AW438849, AA743021,


AI708566, AA045663,


C05053, AW268698,


AW078896, AA312059,


AA894905, AA586355,


AA947891, AA724345,


AI024387, AI631228,


AA878972, W38328,


AL048109, AA125949,


T59457, F13274,


AA172290, AA831417,


AI905071, AA047840,


H06542, AA053980,


N31289, AA135017,


AW337421, AA373169,


H06484, T06616,


AA837059, T77300,


AW295581, 239935,


AA759329, N31083,


839702, F10872,


AA057237, T36230,


H89228, 243869,


AA598442, AA233777,


AA056096, D53439,


AA035459, AA484066,


AA018173, 896192,


AA693386, AL048108,


AW392317, AI090106,


AA732389, AW150491,


AI270737, 838185,
237004,


AA501472, AA483236,


T19033, AA749100,


W04596, H41069,


AI200780, AA580553,


AA405183, T18978,


N56489, AW189389,


AA079574, D19937,


AI9051 O 1, AR044461,
and


186


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF061739.


HE8DL23 41 693641 1 - 487 15 - 501 W78996, AW172850,


AW169307, AW401638,


AA399131, AA251893,


AI361767, AB015318,


AF068706, AF068707,


AL135999, and AL135999.


HAJCL60 42 1218435 1 - 19881 S - 2002AA626698, AW004763,


AL042663, C00237,


M13442, M13443,


AR051318, K03460,


AF172400, AR023871,


A76335, 583440, X52308,


U79414, AF026124,


AR038854, AF017790,


X59813, AF000167,


AL096720, AL133655,


E01314, and AF078852.


HNGJI55 43 722240 1 - 290 15 - 304


HSDIF59 44 1207943 1 - 13631 S - 1377


HKABW26 45 1127499 1 - 776 15 - 790 AI801903, AI768712,


AI333177, AI805900,


AA588685, AW294927,


AI492047, AI302255,


AW166321, AA973387,


N22140, AA808664,


N71516, AA215502,


AA029037, AA099693,


AA927014, AA099692,


AA768510, H47467,


AW291894, AL039390,


AL046681, AI890907,


AL046137, AI926046,


AI049726, AI249936,


AA598862, AL042009,


AI672187, AL042551,


AL047188, AA446038,


AL044204, AI926327,


AI920975, AL045166,


AI446483, AL120789,


AL045503, AW337394,


AI888581, AL043088,


AL134357, 299289, and


AF201334.


HE9HE89 46 1189795 1 - 211815 - 2132 AI801903, AI768712,


AI333177, AW020553,


AA621052, D60213,


AA310511, AI805900,


AW294927, AI613127,


187


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI492047, AI302255,


N51701, AW166321,


AA973387, AI908563,


AI525785, N48792,


AI719605, AA588685,


AI077404, AI243385,


AA573234, AI284732,


AA345848, AA808664,


AA587950, N71516,


AA278670, AA215502,


D60202, N22140,


AA278202, AA327840,


AI080061, AA029037,


w AA099693, AI862953,


D60212, D60201, N45583,


N64148, AA099692,


AA927014, AA768510,


H47467, AW291894,


AA446038, AF201334,
and


299289.


HEON059 47 741361 1 - 848 15 - 862 W37916, AW369323,


AL138197, AA292636,


AA789205, AA278688,


AI077497, AI670821,


AW 117287, AA608906,


AA382777, AA724269,


AA278680, AA827227,


AI160796, AA600253,


AI990171, AA625768,


W37874, AA421286,


AA292637, AA844108,


AA917528, AW316905,


AA768446, and AF117210.


HE6BQ76 48 775616 1 - 353 15 - 367 AA099543, AA669197,


AA127290, 818710,


H08922, W79474,


AW 118919, AW304022,


N41498, AA213595,


W86555, H51174, 225317,


AA304745, AI609937,


H57648, AF083033,


AR028451, AF072860,


284477, and AF083032.


HDPYE27 49 1216489 1 - 2302 15 - 2316AL134742, AA778440,


N37091, N75091,


AA639654, AW022145,


AW007159, AI683811,


AA053789, AI122783,


AI744925, AI467948,


188


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
N50027, AA057679,


N77557, AI445747, N28928,


AA243776, AI363334,


AI336372, AI963359,


AI040028, AI479455,


AA165101, AW385014,


AI500179, AI697381,


W37774, AI339917,


AI653997, AA693500,


AI806196, AA228021,


AI333501, AI142930,


N21339, W60875,


AI431532, AA035737,


AI632712, AA063622,


W73050, AA975536,


AA576042, AI124024,


AI658995, AI440474,


W60732, AI034259,


AA975579, AI311527,


AI969455, AI080681,


AW025344, N64476,


AI887691, AA926635,


AA281420, N35915,


AA278973, AI346272,


N62274, AA410201,


N67994, AA543095,


N27998, AI394262,


AA281380, C75130,


AA582148, AW341214,


AI061278, AI370984,


N43014, AI147848,


AI580843, AI983578,


896110, N75197,


AA232786, W37990,


N95298, H97841, AI074120,


N36648, AI282107,


AA233347, 856367,


D56390, N36876, H04632,


N33506, AA130092,


AI797419, AA974076,


H42814, W80686,


AA766923, AI748830,


H21609, AI917261,


W80865, AI453342,


832872, 831373, AI095232,


AA912662, AI915416,


M78700, AI002766,


W44789, W29011, N52321,


AI077579, AA314971,


189


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
N22321, N33338, AI026003,


AI830057, AW021634,
r


AI299595, AI246278,


AA558723, AA847542,


AI191815, AI765002,


859115, AA743613,


AA730188, AI302598,


AI244544, H12348, T08244,


AA094999, T16664,


AI421363, N47433,


AW370192, N33351,


T34113, W07579,


AI873477, D58486, T30904,


859114, AA814563,


F06223, AA776919,


AI274376, C04716, N46147,


AA665858, AW149733,


AA573244, N43000,


AA298197, T92184,


F12336, AA386234,


AI825897, AI333196,


C03677, T66084,


AA228112, C05913,


AA854336, 239132,


896072, 833003, N80416,


878803, AI094600, F06176,


AI379933, 824775,


AI391739, T87636, 876203,


AA737626, AA082696,


AA302875, AI698554,


T31494, T56448,


AA296902, 242327,


AA725147, AA370253,


AA290935, 224957,


T09018, T30823, 239088,


T08243, AA285111,


AA861783, AW 193046,


H72982, AI127992, H89910,


AA298178, N46405,


AA243648, AA493205,


AA410182, H89911,


N42305, N87136, N62343,


228664, AI828386,


W24772, N80865,


AW386714, AI886156,


835868, N92210, F13786,


AI866424, T35613,


AA384765, AA054617,


AA877058, D56703,


190


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
N26016, N43789, T92218,
838141, AW380302,
AA300758, AI735379,
.
T31648, T35315,
AA096136, 833002,
AA486303, F05590,
w AA310870, 838053,
D82417, 835869, N38773,
F01856, AA036707,
T34827, T30754,
AA809971, AI244054,
H72981, AF062347,
AF062346, AF062072,
AF062071, AL031779,
X75687, L37047, 577728,
and S77733.


HDPCN94 50 1216484 1 - 2809 15 - 2823 AI094945, AW338968,


AI126294, AW026228,


AI982584, AI589050,


AI309065, AA026692,


AI018447, AW003916,


AW338195, AA424111,


AI279956, AA400155,


AA952950, AA939286,


AI018115, AA400313,


847388, AI290258,
N93448,


D79950, W21217,


AI493787, AI263862,


AI338763, AI042507,


AI619662, AI567582,


AI620302, AI671642,


AW169653, AI309401,


AI499890, AI524654,


AW059713, AI800464,


AL037558, AI627988,


AI521005, N42321,


AI886415, AL036673,


AW151136, AL119863,


AI783530, AW020693,


AI254226, AI889168,


AL079963, AA494167,


AA287231, AI890507,


AL036631, AW163823,


AL079960, AI267162,


AW084447, AL038779,


AW074993, AW072484,


AI349614, AI573026,


AW193134, AW071380,


AI436429, AI273094,


191


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI282355, AI349256,


AI312152, AI307557,


AI345347, AA833760,


AI620093, AW075084,


AI687168, AI349937,


AW021373, AW302992,


AI345688, AI334884,


AI307543, AW051059,


AW071412, AI307708,


AI312325, AI340659,


AI687166, AI336633,


AI345567, AI334930,


AI309443, AI307520,


AW 169604, AI340664,


AI349957, AL040241,


AI345739, AI312143,


AI345005, AI446373,


AI310927, AI589668,


AA640779, AI307578,


AI349955, AI580674,


AW075093, AI494201,


AI345143, AI312357,


AL119791, AL041150,


AW302965, AI254727,


AI343112, AI874166,


AI473451, AI582932,


AW074869, AW 167918,


AA417129, AL036274,


AW301300, AI349598,


AA572758, AI538764,


AL036664, AWO75207,


AI343037, AW 161579,


AI345735, AI312428,


AW085786, AI866798,


AW022682, AI431424,


AI500714, AW023338,


AI358701, AW073898,


AA420722, AW105601,


AI284517, AI923989,


AI572717, AW080995,


AI307210, AI567612,


AI648684, AI648567,


AI433034, AI440263,


AI589267, AI310575,


AI313320, AI313352,


AI340533, AI349266,


AW072719, AI312146,


AI312339, AI309431,


AI345258, AI690748,


192


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI251221, AW 161156,


AI590686, AI281757,


AI783504, AI311604,


AL048656, AI923370,


AW089572, AW071417,


AL047422, AI349269,


AI433157, AI702073,


AI310945, AI539153,


AI682593, AI340627,


AL120853, AL036980,


AW 130863, AI630747,


AI345224, AI633125,


AI698391, AL046595,


AW083778, AI311892,


AI919593, AA983883,


AI889147, AA613907,


AA580663, AI670009,


AI889189, AI917963,


AI288285, AI890806,


AW196105, AW269097,


AI955906, AI918449,


AI872910, AI310925,


AI587121, AW163834,


AW072588, AI312399,


AW162194, AI933992,


AI537677, AW263804,


AI344826, AW268253,


AI345156, AI690946,


AI554821, AI348854,


AI307569, AL036802,


AI311159, AW079334,


AI336495, AI336654,


AI554343, AI567351,


AL036396, AI382670,


AL036403, AI950664,


AL039086, AW 152182,


AA012905, AI307736,


AI345562, AI345026,


AI307454, AL038463,


AI348897, AI800433,


AF120323, AL122086,


AF120324, AB022915,


AF121102, AF195656,


I48978, A08916, A18777,


I89947, A08913, A08910,


AR038854, A08909,


A08912, A08908, I89931,


I49625, U42766, I89934,


I89944, AF141289, A08911,


193


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
X52128, A08907, U49908,


536676, AL133557,


AF 118094, I48979,


AF067790, E05822,


AF106862, Y09972,


A03736, AF106657,


A90832, AJ238278,


AL133560, U00763,


Y11254, AR068751,


U87620, 297214, I03321,


AL117416, AL133113,


Y 10080, AL080127,


AL049465, X70685,


X80340, AF111112,


AF091084, AL122050,


AL137550, AL137463,


AF113694, X87582,


AF100931, AL049283,


U35846, I96214, AR034830,


AL133568, AF162270,


U80742, AL110222,


AF118064, AL049466,


AL137554, AF061795,


AL122110, AL137271,


AF151685, AL122106,


AF146568, AF090943,


AL122093, AL117583,


AL050277, AF113690,


AF199509, AR013797,


A77033, A77035,


AF017152, AF097996,


AL137560, S78214,


AF158248, E12806,


Y07905, AL137660,


AL049464, E02349,


AC004227, I00734,
Y 16645,


AL080086, AF067728,


AF028823, AF090900,


I26207, AL049452,


AF113676, AL049430,


AL137705, X96540,


AL137530, X63162,


AL050024, AL050116,


M86826, AB007812,


AL137429, X82434,


AF090934, A07647,


ALl 10225, U91329,


AL080124, AL080163,


AF106827, AL110280,


194


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AL122098, AF090896,


AL117649, AL110171,


AF026816, X57961,


X84990, AJ000937,


AL049300, U68233,


561953, I92592, AF078844,


U68387, AF090886,


S69510, AL133075,


A76335, AL137526,


L19437, S76508, AL133080,


I41145, U58996, U78525,


AL133565, AL133606,


AL137521, AL133640,


AL137539, AF090903,


AL050149, X98834,


AF126247, AL117460,


AF087943, AR038969,


AF051325, AF031147,


AL137459, U72621,


AF079763, AJ242859,


AL122104, AF113691,


AF017437, AL122049,


AL117457, AL133665,


AL117435, AL137478,


I09499, AF125948,


AL080159, Y10936,


U95114, E00617, E00717,


E00778, AL133098,


AF113013, AR029490,


AF118070, AL137658,


U49434, AL133016,


AL023657, M30514,


X65873, L31396,


AF026124, AF113699,


AF079765, AL137529,


AL080060, AL050108,


AL137527, A65341,


AL050138, AL133014,


X93495, AL137300,


AL137548, AL117394,


A21103, X92070, E15569,


AJ003118, A15345,


AF113019, AF177401,


AL137283, AF061943,


AR020905, AL080140,


AL110196, Ak'137367,


A45787, AL133104,


AL080158, AL137533,


A18788, AL050015,


195


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AR011880, AL137556,


AF030513, L31397,


AF132676, AL137712,


AF111849, A93350,


AF113677, AL137558,


AF061836, AF032666,


AL122111, AF012536,


AB019565, D83032,
and


237987.


HDPDH64 51 796509 1 - 506 15 - 520 AI929457, AW249044,


AI739490, AB020706,


X14972, X53773, X14971,


and 266177.


HFIJC31 52 828148 1 - 519 15 - 533 AA885328, N79858,
and


AI184184.


HACCH94 53 847143 1 - 1399 15 - 1413 AI093369, AW292321,


AA972431, N40174,


AA746376, AA130392,


AA286750, AA287684,


871586, 871568, 871587,


H03136, H03946, 871567,


AI471079, H97311,


AA365025, AF039686,


AF118670, AR034800,


AF081916, AL161458,
and


AL161458.


HSLAE47 54 1158024 1 - 497 15 - 511


HTEED80 55 849075 1 - 1299 15 - 1313 W87655, AA465429,


AA203572, AI479983,


AI540082, AA192438,


AA836236, AA465358,


AA829419, AA507269,


AW290970, AI796504,


AA904433, AA368409,


AA688079, AW189971,


AA601527, AA393948,


F36549, W87656,


AA320120, AI862710,


F30605, AA196706,
and


AJ242978.


HNTBH68 56 1109052 1 - 579 15 - 593 AW157233, 243649,


C15376, and 820382.


HAJBU67 57 1186051 1 - 1680 15 - 1694 AA436974, AW301595,


AI338889, AI627769,


AI148986, AW295167,


AI095891, AA228704,


AW300645, AA938998,


AW290959, AI584103,


W51788, AW239035,


196


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA631562, AA314478,
T30453, AA593364,
AA380939, AA593259,
D20778, AW 148377,
T19553, AI371361,
AA380937, AA228703,
T19552, AW156939,
AI696364, AF132951,
and
AW514097.


HE20I42 58 857135 1 - 160915 - 1623 AW179274, AL037345,


T60653, AA297223,


AI222285, AA180256,


AI470271, AI569173,


AA654529, T57755,


AW374056, D26549,


X78479, U04354, Y13971,


and AC005281.


HISAR24 59 1206561 1 - 143415 - 1448 AA778721, AI309326,


AI923088, AW157189,


N21676, AW009559,


AW277241, AI015567,


AA142926, AA935517,


N71214, AI340068,


AA046446, AI890415,


AI760248, AI421490,


AI423473, AA576688,


AI342399, AA946956,


AA826534, AA143149,


AA594414, AA233629,


AI803454, AA125818,


N2201 l, AA313266,


AI953431, AI862039,


AW148924, AA315935,


AI245757, AW374039,


N80237, AA488928,


W33046, AW304989,


AA782164, H89121,


T78059, AI344475,


AW403205, AA074821,


AI148049, AI707964,


AA172038, AA864308,


AA702036, AW 163161,


AA034997, AA058701,


AW057651, AA045662,


AA084570, AW438849,


AA743021, AI708566,


AA045663, C05053,


AA312059, AW268698,


AW078896, AA894905,


197


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA724345, AA586355,


AA947891, AA878972,


AI024387, AI631228,


AL048109, F 13274,
T59457,


AA831417, AA172290,


AI905071, H06542,


AA125949, AW337421,


H06484, T06616, N31289,


AA135017, T77300,


AA837059, AW295581,


AA759329, 839702,


239935, AA056157,


F10872, AA405935,


w W38328, AA057237,


H89228, AA233777,


AA598442, AA056096,


D53439, AA035459,


AA373169, AA484066,


896192, AA693386,


AL048108, AI090106,


AA732389, AI270737,


AA047840, AW392317,


AW150491, 838185,


237004, AA501472,


AA483236, AA749100,


H41069, W04596,


AI200780, AA053980,


AA580553, AA405183,


T18978, N31083,


AW189389, D19937,


AI905101, N56489,


AA079574, AI002285,


AR044461, AF061739,


A30543, I19505, U96138,


AL137283, and AF069506.


HMEK039 60 1228120 1 - 2051 15 - 2065AI832149, AA253498,


AA927669, AW 136320,


AA284897, AI375638,


AI141878, AA410733,


AA724418, AI656580,


AA626359, AA633990,


AI341987, AA210941,


AA480438, AI499844,


AI498056, AW135997,


AA595691, AA209463,


AI804771, AI278733,


W52189, AI199374,


AA602519, AA253394,


AI189792, AI151483,


198


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI242359, AI278938,


AA456100, AI831279,


AA455603, N51328,


855869, H22614,


AA496421, W96552,


T16865, 890921, 887952,


AI913942, AA284720,


AA904546, 855788,


887953, 825653, T16864,


H20796, 890911, AI673432,


AI954640, 890922,


AI631375, AI380914,


AA969376, AI123164,


AI355743, 827502,


AA338810, and


AW243972.


H7TBC95 61 865922 1 - 692 15 - 706


HPMBZ21 62 867222 1 - 550 15 - 564 AC005281, X78479, and


D26549.


HLHTE91 63 789603 1 - 1196 15 - 1210AI124644, AA256351,


AA294967, 871807,


AA305696, AW276058,


AI631672, AI861834,


AI888075, and AB020698.
.


HTEPX32 64 1134915 1 - 1348 15 - 1362AI217947, AW237109,


AI918745, AI968403,


AA934788, X84693, and


AW594564.


HE8AM04 65 871156 1 - 506 15 - 520 AA378845, AA332652,


AA331633, AL031774,


AL031774, AL031774,


AL138825, AL138825,
and


AL138825.


HIBEF26 66 871533 1 - 492 15 - 506 AA351087, AA339704,


T31212, 241917,


AW249404, C15783,


C 1 S 823, C 15142,
C 14979,


299716, AF 111179,


AF 111180, AF 104411,


AF 111181, 299716,
299716,


and 299716.


HAOAE45 67 1114497 1 - 1118 15 - 1132AA481981, AA482086,


AA354345, AW440413,


AI679439, AA214170,


AI868537, AA090435,


AW089582, W02284,


AA313857, 298048,


AF031380, and AF038172.


HSDIT49 68 1169473 1 - 940 15 - 954 AI526055.


199


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
HTLHP64 69 1189793 1 - 1383 15 - 1397AI290476, AI811075,


AW070435, AA708916,


AA877889, AA243884,


AI673586, AA535517,


AI682428, 855643,


AI240304, 855421, H42362,


AI446297, AA243877,


AI784582, AA514267,


W05138, F16063,


AA502383, AA502905,


AA862373, H96943, and


N75418.


HETKT65 70 1110514 1 - 803 15 - 817 AL042941, AW408069,


T17359, M78292, 818937,


AW246248, and AL117408.


HOHBY04 71 888190 1 - 725 15 - 739 AW237906, H63227,


W88522, AL133245,


Y17267, AL133245, and


AL133245.


HTFNP84 72 909687 1 - 2474 15 - 2488AI916675, AI823992,


AW082308, AI816135,


AI589007, AI566535,


AW272765, AA766315,


AW242239, AA279943,


AI816094, AI014927,


AI038579, AA578848,


AI476548, AI354483,


AA973322, AA992180,


AA172248, AA279942,


AI392988, AA327978,


AA769228, AA506076,


AA301103, AI653752,


AI370562, AA343765,


N85422, AI540751,


AI282882, AA506075,


AL137710, and L11316.


HDQGZ78 73 1091628 1 - 1613 15 - 1627AW084116, AI453760,


AA412504, AA921849,


AW083007, AA412618,


W93897, AW419472,


AW276980, AW 196340,


AA516262, AA952969,


AI478774, W93857,


AI276233, AA084469,


AA084475, and AF038388.


HHEMD52 74 909742 1 - 1605 15 - 1619AW341445, and


AA885447.


HODFD73 75 1171995 1 - 684 15 - 698 AL050332, AF048976,


~ ~ ~
AF050183, AF058790,


200


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF058789, and AB016962.


HIBDE74 76 1081188 1 - 1496 15 - 1510AW440521, AF114028,


AI475567, AA448233,


AW297752, W93994,


AW235968, AA297792,


AI802944, AA448377,


H81801, H81802, AI864036,


AA350909, AA837157,


AW015303, and AL035106.


HTAIF22 77 910040 1 - 452 15 - 466 Nov 22 2000 3:31:07:
and


450AM.


HUVFZ43 78 910860 1 - 1436 15 - 1450AL121363, AI569727,


AL121364, AA296414,


' 217339, and AA345259.


HNTMB90 79 910934 1 - 718 15 - 732 299396, AL038837,


AL037051, AL036725,


AA631969, AL039074,


AW392670, AL036418,


AL039085, AL039564,


AL036858, AL039156,


AL039108, AL038509,


AL039109, AL039128,


AL036924, AL037094,


AL039659, AL038531,


AL036196, AL039625,


AL039648, AL045337,


AL036767, AL119497,


AL037082, AL037526,


AL036190, AL038447,


AL119483, AL037639,


AW372827, AL039678,


AW363220, AL039629,


AW384394, AL119457,


AL039423, AL036238,


AL039150, AL119319,


AL040992, AL119324,


AL042909, U46350,


AL038520, AL119484,


AL119391, AL037077,


AL119443, AL119522,


U46351, AL119355,


AL119363, AL037726,


U46341, U46347, U46349,


AL134533, AL039410,


AL038851, AL119341,


AL134531, AL119335,


AL 119418, AL 119396,


AL039386, AL036998,


AL036733, AL119496,


201


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AL037615, AL119439,


AL036268, AL037085,


AL 119401, AL 119444,


AL037205, AL037027,


AL037178, AL045353,


U46346, AL036679,


AL042614, AL036765,


AL042965, AL042975,


AL134528, AL134538,


AL042984, AL036191,


AL119399, AL134920,


AL042544, U46345,


AL036719, AL043003,


w AL043019, AL042542,


AL042551, AL042450,


AL043029, AL134542,


AL134532, AL037021,


AL037054, AL036836,


AL036158, AL119464,


AL036774, AL036886,


AL036999, AL036964,


AR066494, ~AR060234,


AR023813, AR064707,


A81671, AR069079,


AB026436, AR054110,
and


AR064706.


HMKCH92 80 910936 1 - 789 15 - 803 T78768, 813299, 814933,


AB003592, D87248,


AC018833, AC034192,


AC026206, AC026206,


AC022381, and AC022381.


HTELV86 81 910946 1 - 1086 15 - 1100AI272244, AA382531,


AI809639, U35371, and


X99043.


HNTAF23 82 910947 1 - 239 15 - 253 AW392670, AL119355,


AW372827, U46341,


AL119483, AL119319,


U46349, AL119457,


AL119324, AW384394,


AL119497, AW363220,


299396, AL134920,


AL134524, AL119484,


AL119363, AL119391,


U46350, U46347, U46351,


AL119443, AL119444,


AI142137, AL119341,


AL134538, AL119439,


AL119522, AL119396,


U46346, AL043029,


202


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AL037205, AL134531,
ALl 19335, AL042614,
AL119399, AI142139,
AL119496, AL134533,
AL119418, U46345,
AL043011, AL042984,
AL043019, AL042544,
AL042896, AL043033,
AL042965, AL042975,
AL042450, AL042542,
AL043003, AL119464,
AL042551, AB026436,
A81671, AR060234,
AR054110, AR066494,
AR069079, and AR043113.


HELHPO1 83 1137839 1 - 1444 15 - 1458AI608791, AA127698,


AI161158, AI354544,


AI084748, AI858808,


AI342497, AA121534,


AW167039, AA534215,


AW087209, AI688929,


AW160657, AW276367,


AA187542, AW276379,


AW276372, AI208010,


AL037531, AI869783,


AA864203, AW022809,


AW163523, AL042389,


N67858, AW014774,


AI128491, AW163251,


AI142006, AI741015,


N71131, W69482,


AL039099, AI283271,


AI246571, AI580768,


AL134219, AA029532,


W69483, N70077,


AW052086, AI929713,


AI354786, AI440409,


AI262591, AI479077,


AA999933, AI692210,


AI591173, AI524321,


AI826162, AI815904,


AA991277, AW029285,


AA315904, AI124609,


AI751795, AW135528,


AI298570, AW406256,


AI950928, AI539728,


AA307850, AI338794,


AA465027, AI970583,


AW156926, AI762457,


203


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA433862, AL041470,


AI928845, AA306587,


AA180001, AA004466,


D51104, AI816418,


AW157249, AA610408,


AW438710, AW 167329,


AA082096, AI279257,


AW242362, AW 162260,


AA088541, AI401664,


AW157417, AW163512,


H22174, AA194754,


W46515, AA304425,


AW439662, AI003392,


w AW247199, H22138,


AI804527, AW405514,


H63206, 846286, H67969,


AW439422, AA907456,


T26421, AA778397,


AI687072, AI815396,


AA325006, AI680477,


AL042390, AW411448,


AA324732, AW 104826,


AW157458, AA187260,


AA083982, AI141702,


AI680118, AW 162409,


AI816045, AW303785,


AA312844, AA730294,


AW406619, AL041471,


AI372525, H80185, T19319,


AW 162596, M62208,


AA009448, H30869,


AL121246, H80342,


H71158, W01561,


AI697194, AW 168145,


AW 162660, AW409733,


H63120, AW088214,


AA325602, M77906,


T19333, AW080823,


886041, W22965,


AA808433, 851405,


AI560028, AAl 13043,


AW 162591, AI538243,


AI970740, AW246768,


AW022184, AA180847,


AA599787, AA434162,


H09396, AW161782,


AA373486, AW003626,


AI672191, AA148746,


AW248562, AW090042,


204


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
U47634, X60786,
AL050056, AF035316,
X79535, X00734, M15052,
J00913, V00389, M11443,
M11442, AF184595,
AF102890, X07011,
U08342, X03369,
AF184596, X60784,
AF120325, M28730,
X04663, AL030996,
S57698, X02344, L08013,
AB011679, X60785,
AF147880, J00316, L06232,
J00315, M28732, M28739,
AF074422, and A74700.


HTLGH72 84 1199896 1 - 900 15 - 914 AA316295, AI018335,


AI453623, AI123197,


AA947467, AA401192,


AI436596, AA749110,


F06683, AA442605,


AA095581, AI972354,


H08256, 855838, AI133156,
.


AI812015, AI334445,


AI499621, AI284131,


AI537677, AI913452,


AI926367, AI174394,


AI523806, AI445165,


AW 149227, AW301409,


AI354998, AL119791,


AI824576, AW004886,


AW161156, AI934035,


AI352497, AL135025,


AL041150, AI648684,


AI312428, AL119863,


AL036980, AI699865,


AI343059, AI224027,


AL040241, AW161579,


AW151136, AI133489,


AL041772, AI349933,


AW163834, AW071417,


AI632408, AW 148320,


AI345608, AI499986,


AI280732, AI349967,


AI636445, AI499285,


AI537244, AI801325,


AI281782, AI500061,


AI345471, AI873644,


AW 168485, AI648509,


F27788, AI567582,


205


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI432040, AI445990,


AI571909, AA640779,


AW163823, AI624548,


AW235745, AI476109,


AI680498, AI478123,


AI690751, AW051088,


AI620089, AI886753,


AI362637, AI500523,


AI340603, AI698401,


AA225339, AI862144,


AW198144, AI433384,


AI446373, AI890833,


AW102761, AI284517,


AI923989, AI521594,


AI950892, AW 198075,


AW168031, AI783504,


AI620284, AI863321,


AW051258, AW080402,


AI628217, AW088903,


AW117746, AI637748,


AI921248, AI611738,


AI275640, AI619502,


AI677796, AI680162,


AI306613, AW268302,


AI802542, AL037030,


AI922901, AI282326,


AI866770, AA449768,


AI250293, AI288305,


AA572758, AW118518,


AI570807, AI933589,


AI635067, AW026882,


AI923370, AI627988,


AI249962, AL079963,


AL038605, AI859880,


AI445992, AL134999,


AI874166, AW 151714,


AI569309, AW 129230,


AI670009, AI433157,


AI696626, AI702073,


AW087938, AI866573,


AW071349, AI684234,


AL121286, AI251221,


AI539771, AL037454,


N33175, AW238730,


AI623941, AL037043,


AL036274, AI797908,


AI500662, 832821,


AI888621, AI539028,


AI273142, AI633125,


206


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI698391, AI915291,


AI678357, AW268251,


AI582932, AL036673,


AI521476, AI679506,


AW079572, AI308032,


AI432969, AI521560,


AI889189, AI308035,


AI284509, AI868204,


AL042745, AA493923,


AI345253, AW088134,


AI783792, AI269862,


AI802826, AL048323,


AI250369, AI310575,


w AI344785, AI345677,


AI888661, AA494167,


AL048340, AW193911,


AW268768, AI498579,


AI963216, AI340533,


AI919107, AI800433,


AI537515, AI569583,


AI888944, AI886123,


W74529, AI524671,


AI570966, H89138,


AL039086, AI475151,


AI358701, AL120853,


AL036736, AL121365,


AA427700, AI249877,


AI251205, AI886181,


AI439717, AI538342,


AI497733, AI814087,


AI251830, AI288285,


AW072719, AI869377,


AI344935, AW068845,


AI687065, AW 130930,


AI270205, AL036396,


AW302965, AI439762,


AI955866, AI933785,


AI520809, N80094,


AW074869, AI554245,


AI633419, AW151785,


AJ242973, U37150, I48978,


AL122098, I89947, I48979,


AF113013, AF125948,


A08916, A07647, A08913,


A03736, A08910,


AL110221, AL137459,


AF087943, I89931, A08909,


AL117585, A74814,


AL137294, I49625,


207


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF113019, AL133075,


AJ012755, AF104032,


AF026816, AL080159,


I33392, Y14314, AF146568,


AL122093, AL133113,


AL122110, AL049430,


X98834, AL137271,


X84990, AL137463,


AL137480, AL122121,


A58524, A58523, E03348,


AF017437, AF067728,


Y11587, AF111849,


AF113676, AF090896,


AL050393, U42766,


AF111112, AR059958,


AL117460, E07108,


AL133565, 561953,


AF097996, ALl 10196,


AL122050, AJ242859,


AF090900, AL133016,


AF057300, AF057299,


AL080060, Y11254,


AL050149, AF177401,


U35846, AB019565,


AL049283, AL133557,


AL050277, I00734,


AF090901, A77033,


A77035, AF079763,


AL137538, A93350,


AL117457, A45787,


AL096744, E00617,


E00717, E00778, AF185576,


AL133560, A08912,


AJ006417, I26207,
X72889,


AF113689, U67958,


AF113699, AL133640,


I42402, AF183393,


AF113691, AF090903,


568736, U80742,


AL117394, AL050138,


AL080124, AL133606,


AF106862, AL110280,


AF113694, A93016,


AR038854, AF118064,


I09360, A65341, AR000496,


U39656, AF210052,


AL133568, A12297,


AL117435, X93495,


AL110222, AL137476,


208


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AL137557, AL049382,


E02349, AL080137,


AF119337, AL137556,


282022, E08631, AF158248,


X63574, AR013797,


AL133067, AF118094,


AF090943, AL049938,


AL137550, AL117583,


578214, AF026124,


AF162270, AL050116,


AF061943, U72620,


AF091084, X82434,


AL050024, AL137478,


AF132676, AF061836,


AL137648, AF017152,


237987, AL050146,


AL117440, AL110225,


AL122118, U00763,


E02221, X96540,


AL122123, AR011880,


AL049466, AL122049,


I03321, AL137526,


AL133093, AF118070,


X70685, E15569,


AL080127, AL133077,


AL133014, AL137527,


AF078844, AL133080,


AL133081, AJ238278,


AF125949, AF113690,


Y16645, AL049300,


AL049452, AJ000937,


AL049314, AF111851,


AF153205, U96683, I09499,


AL050108, X65873,


AF079765, AF003737,


AF090934, AF113677,


AL049464, AR038969,


X87582, E04233,


AL137560, L30117,


AL133072, AL137521,


AF061573, AL137292,


AL133104, L19437,


AL050172, U58996,


AL133098, X92070,


L31396, L31397, E08263,


E08264, E07361, E05822,


Y09972, U78525, U91329,


AL133665, AL080074,


AL137533, AF067790,


209


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF028823, X62580,


272491, A90832, M30514,


AL023657, E12747,


AF081197, U68387,


Y07905, AL117432,


AF008439, AR020905,


AF106827, 579832,
E06743,


AL122111, I41145,


AF022363, AL137523,
and


AL110197.


HHEND45 85 1128226 1 - 582 15 - 596


HMTAY52 86 1137641 1 - 2174 15 - 2188 AI700520, AW149641,


AA861402, AW438617,


w AW 136764, AA846335,


AI884663, AI076276,


H93328, AA813373,


AI093740, H02724,


AI191231, AA725037,


W02044, 896371,


AA317716, AW 188393,


AI422301, AW193480,


AA341954, AI863986,


AA341953, AA628360,


H93832, 896413,


AA322251, H62043,


AA596074, H90193,


879366, AA128507,


879365, and U61836.


HUCPS03 87 1158484 1 - 1366 15 - 1380 AA350185, AI635570,


245871, AA424391,


848915, AA165465,


T77152, T84026,


AA299342, T81218,


AW303853, AC005585,
and


AC004997.


HNHNP81 88 928378 1 - 604 15 - 618 D51060, C14014, D58283,


AA305409, D80253,


D80024, D80166, D59619,


D80210, D80240, D80366,


AA514186, C14389,


D80043, D81030, D80133,


D80247, D59859, D80212,


D51799, D80164, D80219,


D51423, D80022, D80391,


D59787, D80195, D80188,


D80248, C14331, D59502,


D59467, D57483, D59275,


D59610, D80227, D81026,


D50995, D80196, D80439,


210


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
D80251, D80269, D59889,


D51022, D50979, D80268,


D80522, C 15076, D59927,


AA305578, D80038,


D80193, D80045, D80241,


AA514188, AW360811,


D80378, AW 177440,


D80302, C05695, C14429,


AW178893, AW377671,


AW375405, D80157,


T03269, C75259,


AW 178906, AW 179328,


AW366296, AW360844,


w AW360817, D59373,


AW375406, D51103,


AW378534, AW 179332,


AW377672, AW 179023,


AW178905, D51759,


AW360841, AW378532,


D58253, AW177731,


AW177501, AW177511,


D80132, D80134,


AW352171, AW377676,


AW352170, AW 178907,


AW378528, AW178762,


AW 179019, AW 179024,


D51250, AW 176467,


AW 178983, AW 177505,


D81111, D59653,


AW 179020, AW 178775,


AW367967, AW369651,


AW 178909, T48593,


AW 177456, AW 179329,


AW178980, AW178914,


AW 177733, AW 178908,


AW 178754, AW 179018,


C06015, AW352158,


AW352117, D80949,


AW 178774, D59695,


D52291, D45260,


AW352120, D59627,


D51079, AW 179004,


AW179012, AW378525,


AW352163, F13647,


AW360834, T11417,


D80064, 221582, D80168,


C14298, AW378543,


AW352174, D80258,


AW 177728, H67854,


211


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
C 14227, AW 179009,


AI525923, AW367950,


AW 178911, AW 177722,


D59503, AI910186,


AW378540, C03092,


H67866, AA809122,


D51221, AW178781,


D58101, AI905856, D58246,


AW 177508, C 14407,


D80228, AI525917, C14077,


AW 178986, AW 177497,


T03116, AI535686,


AI535850, D59317, D51213,


w D80014, D59474,


AW177734, AI525920,


D45273, AW 177723,
'


C 14973, C 14344,


AW378533, AA514184,


D59551, C14957, AI525215,


D60010, AI525235, D60214,


AI525227, C 14046,


AI557774, AI557751,


AI525912, T03048,


AI525925, D51097,


AI525242, AA285331,


AW378542, AI525222,


AW378539, 230160,


C16955, C05763, 233452,


T02974, D51053,


AW360855, H67858,


AI525237, C04682, D50981,


T02868, C13958, AI525928,


D80314, AI525238, A62298,


AB028859, AR018138,


AJ132110, A62300,


AR008278, A84916,


AF058696, A82595,


AR060385, AB002449,


D89785, X67155, Y17188,


A94995, D26022, Y12724,


A25909, A67220, A78862,


D34614, AR008443, I50126,


I50132, I50128, I50133,


D88547, AR066488,


AR016514, AR060138,


A45456, A26615,


AR052274, X82626,


Y09669, A43192, A43190,


AR038669, AR066487,


212


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
A30438, I14842, AR025207,


AR054175, D50010,


Y17187, AR066490,


AR008277, AR008281,


A63261, I18367, AR008408,


AR062872, A70867,


ARO 16691, ARO 16690,


U46128, D13509, A64136,


A68321, AR060133,


AB012117, I7951 l,
X68127,


U79457, AF123263,


AR032065, 282022,


A63887, and AR008382.


HFIDL68 89 928475 1 - 516 15 - 530 AI375172.


HTLAB16 90 1144562 1 - 1215 15 - 1229AI743990, AI589677,


AW 117688, AA418209,


AA293017, AA393552,


AI671530, AA969969,


H05646, AA868456,


AA836313, 834733,


AW263156, AW206863,


872891, H06920, AI638884,
.


238689, AA280165,


H05647, AI424304,


AI399693, AI685889,


242496, 849606,


AA552211, AW237268,


AA400796, AA621751,


AI468780, AA566051,


D60125, AW237328,


AA651719, AA280537,


AA628052, AI621125,


AI365113, AI590631,


873367, and AW370133.


HOEEU57 91 1158765 1 - 467 15 - 481 AI831578, AA862453,


AI276148, AI925229,


AI344497, AI343937,


AI923983, AI299183,


AI086925, AW387023,


AW387014, AW386986,


AA333257, AI991662,


AW374923, AI093616,


W02289, AA528079,


AA455633, 858057,


AA767913, 821305, and


818269.


HNHCP79 92 565781 1 - 288 15 - 302


HHSDL85 93 942246 1 - 760 15 - 774 812427.


HWADD57 94 943039 1 - 996 15 - 1010AA320236, AC011492,
and


213


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AC011492.


HFKKN77 95 943757 1 - 719 15 - 733


HBGMZ39 96 947112 1 - 588 15 - 602 AI820661, AI791493,


AA989356, AI791282,


AI732537, AI792053,


AW207804, 822360,


872427, AA505927,


822019, 872474,


AC008537, AC008537,


AC008537, AC019337,


AC019337, and AC019337.


HTTCB17 97 1180942 1 - 215815 - 2172 AW070938, AA984061,


AL041636, AL043672,


AI499419, AA533251,


AI820049, AA236580,


AA234444, T64870,


AA761405, AA688276,


H58691, AL042008,


AA421748, 858865,


AL042032, AA157675,


T40471, AA521150,


H23351, AF012373,


N53133, T67438,


AA284019, AL043673,


AA224471, T39198,


AA829259, T99670,


H23240, AA215781,


AA285153, AI039274,


AA056347, AI025833,


AI698363, T99070,


AL046440, AL046465,


T69471, AA652360,


AI090248, AW090185,


AW265609, AI220192,


AA626470, AA428563,


X84692, AC007172, and


U07155.


HEQAP17 98 949358 1 - 807 15 - 821 AI131555, AI769466,


AA215577, AW190975,


AA258335, AA258499,


AL044652, 563848,


Y17793, and A49045.


H2LAD53 99 952181 1 - 348 15 - 362 AA313893, AA332909,


832396, and N57638.


HAMFD12 100 1149676 1 - 320615 - 3220 AI869315, AI795815,


AA147088, AI421559,


AW131473, AA307132,


AI860630, AI859194,


AA554706, AA779596,


214


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI817963, AW 131732,


AI131006, AI131235,


AI298506, AI903044,


AW360926, AA132115,


M79142, AW085550,


832144, AA996236,


AA827499, AA325898,


AI864171, AI810869,


H19200, AW083177,


AI279054, AI683076,


849059, 832143, AI682910,


N48917, AA322186,


W07596, T32822,


AI951120, AA902240,


AI909782, AI302680,


H03637, AA310842,


AA031941, F06992,


AA555053, AI909807,


AA557656, AI874400,


AA319725, AA147145,


AW020184, AA318860,


AA327450, AI935791,


AI702044, AI913289,


AA324945, 849185,


AA132349, AI948650,


833114, F03268, AI419183,


N46415, T29712, AI940337,


N20886, H19201, AI539258,


AA904255, D58578,


AI356503, AA322799,


AA348452, AA348337,


M86105, 862224,


AI636387, AW166586,


833262, 834655,


AA375538, 864601,


AW016275, AA581451,


AA357950, N78707,


AI915762, AI540261,


T12586, AA985516,


AW169409, AL118620,


H03636, 821586,


AA319821, AI962093,


T12578, L07924, L07925,


U14417, AC000395,
and


AW515881.


HE2SY09 101 1186416 1 - 765 15 - 779 AW249044, AI929457,


AL042867, X14971,


AB020706, and AC006942.


HWLLB11 102 954849 1 - 731 15 - 745 AI745636, and AA102414.


215


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
HNTEF53 103 954852 1 - 2342 15 - 2356AA557324, AI655577,
AI696732, AI923200,
AA863360, AW262723,
AI697332, AW275990,
AI436648, AW276183,
856515, AI362521, 853456,
853457, D62878,
AA337301, AA652746,
AW264444, 856123,
AA319338, D79346,
D79250, N56346,
AA886832, and AL138223.


HNTND64 104 954871 1 - 392 15 - 406 AC025090, and AC025090.


HFPFA83 105 955614 1 - 723 15 - 737 C14389, C15076, D59467,


D58283, D50979, D80522,


D80164, D80166, D80195,


D80043, D80227, D81030,


D59275, D59502, D80188,


D59859, D80022, C14331,


D51423, D59619, D80210,


D51799, D80391, D80240,


D80253, D80038, D80269,


D59787, D80193, D59610,


D80212, D80196, D80219,


D81026, D59927, D57483,


D80378, AW177440,


D80366, D80251,


AA305409, AA305578,


D59889, D50995, D80024,


D80241, D51022, D80045,


C14429, D51060, C75259,


T03269, AW 178893,


AW 179328, AA514188,


AW378532, D80248,


C14014, AW377671,


D51250, AW369651,


AW 178762, AW 178775,


AW177501, D80134,


AW177511, AA514186,


D80133, AW176467,


D58253, AW360811,


AW352117, C05695,


AW375405, AW352158,


D80268, AI910186, D80132,


AW366296, AW 178906,


AW360844, AW360817,


AW375406, AW378534,


AW179332, AW377672,


AW 179023, AW 178905,


216


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
D80302, D59627, AI905856,


r
AW378540, AW352171,


D80258, D80439,


AW377676, AW352170,


AW 177731, AW 178907,


AW 179019, AW 179024,


D59373, D80247,


AW 177505, AW 179020,


AW360841, AW178909,


AW 177456, AW 179329,


AW 178980, AW 177733,


AW378528, AW178908,


AW 178754, AW 179018,


AW352174, 221582,


AW360834, D51103,


AW 179004, AW 179012,


C06015, AW 178914,


AW378525, AW367967,


D80157, AW177722,


D51759, AW177728,


AW179009, AA285331,


AW178774, AW178911,


D51097, AW378543,


AW352163, D58101,


D80064, D58246, D80014,


D59503, AW178983,


AW352120, AW178781,


T48593, AI535850,


AW177723, T11417,


D59653, AA809122,


AW 177508, D45260,


D59317, C 14975,


AW378533, AW367950,


F13647, D81111, H67854,


C03092, C14227, H67866,


AI557774, AI525923,


AW 177497, T02974,


AI557751, AW178986,


T03116, C 14298, D45273,


D52291, AW177734,


D59474, AI525917,


AI525227, D59695, D60010,


C14973, AI535961, C14344,


C14407, AI535686, C14957,


D51221, D59551, AI525920,


AA514184, AI525242,


D60214, T03048, C14046,


AI525912, AI525235,


C16955, AI525925,


217


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI525222, D80168,


AW378542, AW378539,


AI525215, AI525237,


C05763, 233452, AI525928,


AW360855, T02868,


D51213, D31458, H67858,


ARO 1813 8, AJ 132110,


A84916, A62300, A62298,


AR008278, AF058696,


AB028859, X67155,


Y17188, D26022, A25909,


A67220, D89785, A78862,


D34614, D88547, I82448,


Y 12724, X82626,


AR025207, AR016808,


A82595, AR060385,


A94995, AB002449,


AR008443, AB012117,


I50126, I50132, I50128,


I50133, AR066488,


AR016514, AR060138,


A45456, A26615,


AR052274, A85396,


AR066482, A44171,


A85477, I19525, A86792,


Y09669, A43192, A43190,


AR038669, AR066490,


U87250, AR066487,


X93549, I14842, A30438,


I18367, D88507, AR054175,


D50010, Y17187, A63261,


AR008277, AR008281,


AR008408, AR062872,


A70867, AR016691,


AR016690, U46128,


D13509, I79511, A64136,


A68321, AR060133,


X68127, AF135125,


U79457, AF123263,


AB023656, AR032065,


AB033111, X93535, and


AR0083 82.


HHAWC08 106 957942 1 - 1870 15 - 1884AI860000, AI984180,


AI052115, AW027098,


AA507892, AI269682,


AI860186, AA910656,


AA911665, AI122607,


AI982553, AI492064,


AI148135, W56156,


218


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
W60937, AA974459,


AI419847, AA009421,


N28887, AI673827,


AI277360, AA740276,


AA778158, AW250214,


AA456771, AI034295,


N35234, AW 170157,


N30305, AW247011,


AW026104, H99254,


AA654123, AA830800,


AI141374, AI074187,


W31386, AA524317,


AI261816, AW403968,


AA846516, N39743,


AA480954, AI280981,


W60872, AW073283,


AA761349, AA683175,


AI207047, AW236672,


AW389139, AA009725,


AW058117, AI073671,


AI092406, AI085232,


AI188288, AA113312,


W15516, AA653970,


AW352169, N95282,


AA551600, W86394,


AI084709, N78792,


AI475205, N42029, N55379,


AW001694, AI355933,


AA 181712, AA004431,


N44897, H94052,


AA699746, T48085,


AA730615, AA112533,


AA306422, AA342623,


838707, H80310, W38668,


H06714, T51462,


AW375770, H06763,


AA302273, F13201,


AA033586, H81887,


W86393, AA687134,


AI269924, AI933743,


889868, AA033585,


AI863793, AA315202,


830828, AA772763,


AA678198, AW375790,


F10804, AW078473,


N26380, H80311, AI159939,


AI383032, AI300606,


AI027171, AA310599,


AA887439, AW403264,


219


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
T75396, AA091936,


889830, N41533,


AA810312, N77721,


N58353, AI768718, 806623,


W37176, H94132, 806681,


AW449471, AA026195,


AA026267, W24414,


AA936706, AI033915,


H81888, W07220,


AA854111, AA826955,


879020, N43834,


AW388723, N77385,


N25010, AA879094,


AA322203, AR009648,


AL096870, AL096870,
and


AL096870.


HBWBF71 107 1189778 1 - 582 15 - 596 AA954402, AA095359,


N88601, AA247964,


N84855, N83168,


AA096046, H58760,


N83991, N88782, N89520,


N83993, AA247827,


N83992, N84048, N84718,


AA095641, AA096066,


N86694, N84830, N55698,


AA471338, N84712,


N88518, N84829, N87989,


N87898, 578798,


AF103726, AF039698,


AF102850, U48696,


AR066487, AF045432,


AF032922, U39066, and


AJ243486.


HTLIT03 108 1152266 1 - 125415 - 1268 AA886893, AA862723,


AA470745, AA992987,


AA928557, AI913313,


AI073998, T81075,


AI277238, T86154,


AI821002, AI821291,


T47170, C00316, and


AC004531.


HSLFH12 109 970661 1 - 418 15 - 432


HE8IVI24 110 971296 1 - 737 15 - 751 AA883367, AA332611,


AA732890, AI283442,


AI673342, AI631153,


AI200800, AI910962,


T11417, D80258, D59503,


D80014, D81111, C14227,


D80064, AI557751, D58246,


220


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
C06015, AA514184,


AI535959, AW178893,


AW 178907, AW375405,


AW 177440, AI535686,


AW360834, AW178908,


AW360811, D80314,


AA809122, D80251,


D80253, C03092, D80247,


D80043, AA285331,


AW 176467, C 143 89,


AW179328, T48593,


AW375406, D80439,


AW378534, AW179332,


D58283, AW377672,


AW 179023, AW 178905,


D59859, D80022, C14331,


D80166, AW177731,


D80195, AA305578,


D80193, D59927, T03269,


D59467, D51423, D59619,


AW378528, D80210,


AW 178906, D51799,


D80391, D80164, D59275,


AW 178762, D80240,


D80038, AW179019,


D59787, D80227,


AW378533, D59502,


AA305409, AW378532,


F13647, D45260,


AW178914, AW378542,


AW360855, AW377676,


I50126, I50132, I50128,


I50133, AF123263, A70867,


D88547, AR062872,


AR066488, AR016514,


A62300, D50010, X82626,


AR066487, Y17187,


AR060138, A84916,


A45456, A67220, D89785,


A62298, Y09669, Y17188,


AB028859, A82595,


A78862, D34614, A94995,


D26022, AR060385,


A30438, AJ132110,


AR018138, A26615,


AR052274, A43192,


AR008278, X67155,


Y12724, A63261, A43190,


AR038669, AF058696,


221


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
A25909, X68127,


AR008443, AB002449,


AR025207, AR016691,


AR016690, and U46128.


HMTAX31 111 1154339 1 - 896 15 - 910 AA410486, AA374753,


AA411633, AW385663,


AA625487, AA256096,


AA411671, N36626,


AW188661, AA406582,


AA847680, N25994,


AW 167272, AW 151243,


AA621715, AA345748,


AA827049, AA284022,


AA815315, AI829183,


AI290229, 870442,


AI040507, H42445,


AI818989, AI040765,


AA812382, AI636434,


AI015551, AW167634,


AI085194, AA983613,


AA406387, AA484968,


AI278749, H82446,


AI342313, AA769207,


AI283824, AA911296,


860575, T53444,


AA463739, AI128463,


AI126964, AI655254,


AA872115, AA410304,


T30540, AA528102,


AA985260, and H42417.


HE8UY87 112 1171975 1 - 1017 15 - 1031AA488339, AA332909,


884768, AA015949,


AA313893, 832396,


832397, and N57638.


HTPDV62 113 1138729 1 - 902 15 - 916 AI183902, AA683089,


AA617873, AA428773,


AA371747, AA302461,


AA490902, AA769215,


AW001581, AI371135,


AA541648, AI369827,


AI190177, AI141079,


AI245438, AI095638,


AW007486, AA888067,


AA534074, AI798016,


AA632013, AI554292,


AI422715, AW170631,


AI003005, AI818273,


AA299236, AI356047,


AI215137, AA126607,


222


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
W58692, AW182892,


AW006997, 854752,


AI971642, AA652728,


AA700953, AA594060,


AI332799, AI991239,


AA464316, AA643082,


AA287161, AI690764,


AI184848, AA534625,


AA482551, AW275871,


W73756, AA482406,


AI312662, AW083880,


AA767390, AI091265,


AA128033, AI499037,


AA029784, AA737008,


AI291962, AA524858,


AA069805, AA305086,


AW173571, AI590872,


AI291963, AW269677,


AW296379, AI497938,


AI092881, AA102025,


AA642110, AA765044,


H56621, AI671631,


AI200177, AI348340,


AA887225, AI573191,


AA470747, AA491087,


AI038522, AA609354,


AA485738, AA284500,


AI302373, AA806051,


AI358650, H19929,


AA829537, AA286924,


AA937034, C00792,


AA723098, AA594909,


AI141128, N66142,


AI833016, AW328470,


AA128009, AA523296,


AA808375, AI199216,


AA761304, AA953934,


AA425912, AI028399,


AI568372, AI202858,


W61335, AA641528,


AI863193, AI347365,


AW170380, AA974521,


N31038, AA706349,


W58693, W76033,


AA306100, AA838347,


AI750639, N20116,
T08092,


AI750622, AA151694,


T52641, AI272226,


AA746219, AI199802,


223


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA399424, AA369769,


U66895, L76200, and


Al 1042.


HMUBV12 114 1227616 1 - 1816 15 - 1830 AI377407, AI591053,


AI140794, AA085250,


AA085176, AA568840,


AW059669, AF036035,


AF040710, U90094,
and


U73167.


HMIAH32 115 1171989 1 - 701 15 - 715 AA890211, AA236800,


AI018543, W15550,


AA815190, AI263837,


AI250936, AA709401,


w AA236846, AI383379,


AA689421, and D87467.


HSIDX67 116 1228968 1 - 1393 15 - 1407 AI634902, AI632135,


AI990924, AA242743,


AW 169040, AA242764,


AW138927, AI184821,


AI200421, AI202175,


AW196380, AA828925,


AW 183069, AI086348,


AA778034, AA580419,


AI350207, AI949886,


AW27511~2, AI871551,


AA927997, AI381743,


AI961077, AA778076,


AA453300, AA453544,


AA167098, AL046926,


AA775150, N99406,


AA452417, AI890702,


AA166911, D14533,


U10347, X74351, S74024,


U16815, U10345, U10346,


U10343, U10344, X74349,


X74345, and 264286.


HMTAV95 117 614936 1 - 395 15 - 409 AB023187, AL137000,


AL137000, and AL137000.


HCE3W04 118 1206659 1 - 2100 15 - 2114 AI677902, AI338780,


AI684570, AI928887,


AW 136644, AW264299,


AI652923, AI089627,


AI298483, AI372875,


AA463846, AI141311,


AW204313, AW 129570,


AI374899, AW 134722,


AW305132, AI366527,


AA463333, U47343,


AA031465, AI654427,


224


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW205086, W26816,


AI217285, D81166,


AA906309, AI382756,


AA031486, D80712,


AA054522, AA325176,


AA884159, AA323357,
and


AA247154.


HMVAC92 119 731732 1 - 454 15 - 468 C18294, AAl 15838,


W76458, T32095, T30710,


AA378552, AW360837,


808104, W23225, T35980,


N45490, AI554624,
T35790,


AI313133, T84898,


AW391980, 220579,


AA357067, T35190,


AA310762, AA328222,


815571, AI205283,


AA035103, AA448769,


AA214347, W38702,


N56257, AA116099,


AW401574, AW411042,


AA371686, AA452303,


AI570120, AA613412,


W61124, AA093588,


T63232, AW368742,


AA868496, AW338692,


AW 167217, AI954547,


AI955639, AA079294,


AA478499, W24470,


870678, AA657656,


AA166897, AA453225,


AA341741, AI697147,


T 16000, AI921707,


AA894909, AA551115,


AF128527, AF126181,


AF128528, U92544,


298046, AB029037,
and


A75460.


HE80U68 120 1086802 1 - 657 1 S - 671 AA446069, AA429913,


AA121710, AW104301,


F07861, and AB002349.


HMUBI80 121 1206766 1 - 2142 15 - 2156 AW299947, AI627241,


AI693196, AI693651,


AI912660, AW 193683,


AI700973, N39338,


AA725613, AI129975,


AI420001, N39022,


AA446088, AA156932,


AI200504, AA156939,


225


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
W72621, AA608671,


W84482, AW071002,


AA429734, AA827783,


AI609691, AA789083,


AI096521, AI807324,


AI741384, AA135719,


AA682230, AI219693,


AI128552, AI457931,


AI183320, AW073830,


AA767680, W67456,


AI146559, AA406621,


AI191714, AA037151,


AA975460, AW236574,


N58764, AI282129,


AI220018, AI040340,


AI765710, N90492,


AA612607, AA877635,


AI422402, AA502264,


AA135877, AA150224,


AA148768, AW195886,


W03532, H98048,


AA722904, AA278951,


AA903213, 878489,


W 19223, AA768151,


AA655014, AI418277,


866829, AI380027, N67714,


T84077, AA973687,


AA569504, 874090,


AA742936, AI242123,


AA027856, H77471,


H97347, H02008, 867927,


AW207778, AA654277,


AA135876, AA688191,


824196, H02106, H77472,


AA216739, AI916580,


866743, N77448, T56468,


H88133, N39327,


AA150679, N45175,


AI652805, AA644283,


AI263823, 831686,


W84328, AI458900,


H88134, N69228, H00833,


874089, AA150756,


H01217, AA906258,


AA569516, 824195,


878529, AA479390,


N45185, AA907738,


T56521, AA903324,


AW371074, AA353909,


226


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
T97790, AA579836,


AA879472, H12526,


AA927935, AA216519,


AA975448, H12473,


827236, T83430,


AA411693, 861052,


827440, AI698993, 869359,


869360, W25349, T97690,


N30828, AA027912,


AI269274, W67457,


831728, AA283743,


AA927936, AI192400,


AL049940, AF179286,


w AB029551, AF101779,
and


AF085840.


HE9RA75 122 766779 1 - 651 15 - 665 AC009648, AC009648,


AP002502, AP002502,


AP000907, AP000907,


AP000788, and AP000788.


HOLTH089 123 1085594 1 - 750 15 - 764 H43782, and AB002378.


HFITE38 124 1063475 1 - 604 15 - 618 AI554624, AI313133,


AW391980, W76458,


AW401574, AW411042,


AA357067, W61124,


AW360837, C18294,


AA852415, T35790,


AL138163, T35190,


T35980, AA331917,


AA115838, T30710,


AA894909, T32095,


AW088409, AI951963,


AI909669, D55453,


AW361222, AI983723,


AI921707, AI205283,.


AA868496, 220579,


AA551115, AI811575,


T08329, AI570120,


AI963063, AI697147,


AA378552, N56257,


AW073182, AA613956,


AA971201, H18459,


AA116100, AA507600,


AI269028, AW338692,


AA736816, AI077651,


AI377484, AA703778,


AI535813, N47602,


AA424243, AW 148920,


W63686, AA765057,


AW166594, AI203352,


227


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA035103, AI354635,


AA749002, AI302562,
r


AA535981, AA451680,


AW163074, AA035501,


AA723482, AA535894,


AI955639, AA909787,


AI092656, AA706448,


AA172257, AA665951,


AI333516, AA478500,


AA371686, AA923643,


238401, T32094,


AA116099, AI204027,


AI890693, N45490,


AA452876, AA452303,


AI928430, N55103,


AI824568, 860321,


AI362729, AA703829,


AA908497, AI207027,


. AA935306, N92292,


AW182581, 815571,


AA719390, AA346276,


AI139827, AI091673,


AA723475, AI040346,


AW050447, AA310762,


AA729774, AI216208,


AI538886, AA853070,


AW078554, AI684984,


AI096337, AI690416,


AA437256, AA916279,


AI745105, AI222527,


AA866024, AI199134,


AI271816, AW 189656,


AI129909, T 16000,


AW072608, AI669857,


AI095643, AI144522,


AI598169, T15580,


AI612922, AI564907,


AA176356, AI263062,


AA016219, AA613497,


AI565670, AI306585,


AI934895, AI337663,


AW001407, AA931614,


AA732764, AA722353,


AA703701, T33343,


AA587632, T31391,


AA643880, T63580,


AA643888, AI917557,


T32270, 822550, 880887,


AA284974, AA478139,


228


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI742813, H13755, 823603,


AA079293, AI675597,


AA662312, AW 167217,


AA613412, AA604206,


808104, AA877259,


AA470818, AA532869,


AA483863, 822551,


AI301879, AA478499,


AA854235, AI354236,


W73618, AW005176,


AA081759, T84898,


AA745131, AA552419,


AI369794, T15699,


AA573836, AA706559,


AI924630, AI688807,


H62299, 895675, AI857403,


T62981, AI954547,


AA400971, 870679,


W23225, AI061326,


AI318066, AA039316,


AW068748, 826158,


AA868790, 842434,


AA706564, AA991528,


AI269944, W72172,


AA654947, AA328222,


T51637, AI933327,


AA401010, T03649,


AA657656, AA781871,


AA113973, H13703,


W68563, AA478138,


AI972887, T91316, U92544,


AF128527, AF128528,


298046, AF126181, and


A75460.


HSXCB49 125 800501 1 - 684 15 - 698


HHFGP83 126 828162 1 - 325 15 - 339 AC026329, and AC026348.


HPLBP54 127 1212679 1 - 1901 15 - 1915C16805, C17480, D58975,


C 18261, C 16945, C
18700,


C 18638, C 18027, C
16784,


C 18260, C 18222, C
17488,


D58770, C16946, C18483,


C17653, C17965, D58978,


C 17490, C 18701, C
17740,


C 18747, C 18449, C
17748,


D58838, C18252, C17885,


C18988, D58702, C18964,


C19012, D58739, C18542,


D58759, C16754, C17095,


C 17755, C 17886, C
16794,


229


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
C18933, D58837, D59219,


C 17939, C 17663,
C 17522,


C 17714, C 17766,
D58840,


D58983, D59184, C16856,


C18711, C17278, C17232,


C17696, C17749, D58550,


C 18624, C 18255,
C 17586,


D58903, D58947, C17836,


C18888, D58967, C18824,


D58566, C17667, D58713,


C 17572, C 18607,
C 18466,


D58779, C 18475, C
17669,


C18611, C18500, D58543,


C17876, D58655,


AA340250, C 16767,


C 18442, C 17741,
C 17818,


C 16785, D58942, C
17390,


C17721, C18910, C18749,


C17700, D58915, D59228,


C 18013, C 17068,
C 17334,


C17982, C17833, C18143,


C18917, D58491, C18541,


C 18469, D58612, C
18074,


C 18717, C 17992,
C 18349,


C16802, D58800, C18889,


D58758, D58968, C17371,


D59152, C17427, C18066,


C 18166, C 17240,
C 17932,


C16816, C17415, C18551,


C 17817, C 18038,
C 17802,


C 17792, C 16893,
C 17705,


C17089, D58946, C18750,


C18287, D59119, D58496,


D58742, D58823, C18292,


C 18347, N71116, C
18696,


C 17432, C 18423,
C 17348,


AA368116, C 17053,


C19078, D58955, D58925,


C 17670, C 18714,
C 18180,


C 17421, C 17769,
C 17767,


C 17239, C 18621,
C 16824,


C 17125, C 18058,
C 18625,


C 16806, C 17265,
C 18663,


C16764, D59000, D58786,


C17950, D58552, C18649,


C 17981, C 19088,
C 17417,


D58653, D59059, D58600,


C 17666, C 18956,
D59075,


C17498, C18610, D58984,


230


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
C18958, C18534,


AA367984, C18674,


C 16864, C 18758, C
17943,


C 18365, C 17999, C
17910,


C17601, C17772, D59187,


C18603, C18568, C17662,


C 17925, C 18425, C
17302,


C18966, C17575, D58593,


C 18090, C 17283, C
16817,


C17790, C18367, D58445,


C 18196, C 18508, C
18676,


D58572, C18643, C18806,


C17158, C16957, C18709,


D58995, D58993, C18380,


C 18477, AI261401,
C 17822,


D58834, C19051,


AA367416, C17873,


C18384, D58907, C17543,


C18463, C17033, D58648,


C18078, C18761, C18506,


C17128, D58682, D59081,


D58675, C19014, D58540,


C17723, C18313, C16871,


C 17516, C 18413, J00118,


V00573, K02401, J00289,


J03071, AF065215,


AF065216, A00469,


V00519, AF121908,


E00009, M15894, L16556,


E00952, A12770, A15072,


A15074, A03992, A03994,


M15895, M38451, L16552,


L16554, E00141, J03756,


I34298, AF006061, L16555,


L16553, M13438, E00974,


V00520, AF006060, I41411,


I02851, V00593, I03501,


E01123, I02448, I13723,


I01079, I03165, A63631,


E15607, E01250, E15608,


E15609, E01249, E00140,


K00470, I02856, E00814,


E00813, E00812, I07973,


I17475, I34299, I03481,


U02293, AF110644, I03167,


I03503, E00002, A00501,


I02588, E01441, A18985,


I34300, I18458, E08200,


E05299, I02855, A10352,


231


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
I21239, A04711, A04745,


A04746, A04712, A17655,


I02858, AF023477, I02857,


M25118, A17660, A17661,


I63120, and AB019574.


HCE4R40 128 858456 1 - 401 15 - 415 AW161406, 890781,


D86957, A87006,


AC004775, and AC005742.


HRABU93 129 1206777 1 - 2267 15 - 2281AW161406, W22101,


AW163611, AW140025,


AA326433, 890781,


853169, AA446363,


U83543, AI380305, H19578,


H23911, H51386, H19075,


H46576, AI609704, D86957,


A87006, AC004775, and


AC005742.


HWLQH41 130 1176226 1 - 1249 15 - 1263AI417842, AI127930,


AI202751, AW237652,


AW241522, AI633241,


AA828649, AI056043,


AI097032, AA595596,


AI332697, AW009101,


AI391721, AI288942,


AI742779, AI741429,


AW338282, AI633779,


AI218345, AI971047,


AI290280, AI754557,


AA812206, AA586780,


AW 117606, AI924489,


AI284061, AI689103,


AW207270, AW294513,


AA568817, AI478517,


AI657130, AI074577,


AI990149, AA806346,


N51855, AI149292, D61975,


AI033281, AA384212,


AI917469, N53363, N73526,


H23985, 828358,


AA677342, AA776965,


AA878338, H91288,


AA748783, H90379,


828562, AA328303,


H22705, AI719957, N54120,


N46676, AA354773,


AI001793, AI553758,


N39234, AF085734,


AJ236876, AJ236912,


AF072521, and AJ007780.


232


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
HMJAH61 131 1176228 1 - 655 15 - 669 AI417842, AI127930,


AI202751, AW237652,


AW241522, AI633241,


AA384212, AI742779,


AI056043, AA828649,


AI290280, AW009101,


AA595596, AI097032,


AI332697, AI391721,


AI288942, AI741429,


AW338282, AI633779,


AI971047, AI218345,


H22705, D61975, H90379,


AI924489, AW 117606,


AI754557, AI284061,


AA812206, N54120,


AI689103, AA878338,


AW207270, AW294513,


AI478517, AA776965,


AI074577, AI657130,


AI990149, N46676,


AA568817, AA354773,


828562, ~N51855, AI149292,
.


AI001793, AA806346,


AI553758, AJ236912,


AJ236876, AF085734,


AJ007780, and AF072521.


HBMXU88 132 1228331 1 - 3849 15 - 3863AW069306, AI743175,


AI803970, AI811472,


AI027704, AA099277,


AI168623, AI276150,


AI824726, AA213703,


AW243339, AA287703,


AA483707, AA287702,


AA213668, AI653168,


AA371310, AI434885,


838844, AA355129,


AA342147, AA342146,


AA365652, AA828028,


AI581083, AA927786,


AA282618, AA099276,


AW364617, AA027167,


AI968421, F24601,


AI913352, AI302397,


AI040349, T56496, 805710,


T83234, AI984941,


AI184494, AA480189,


AI128765, AA027168,


AA382209, AI935351,


AB023172, AP000245,


233


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AP000127, AP000205,
AF143869, and AW572672.


HNTCI60 133 1223477 1 - 3693 15 - 3707 AL046015, AA768846,


AI671768, AI961239,


AI884699, AA514474,


AI521280, AI139530,


AA156958, AI679628,


AI885479, AI336225,


AI735075, AI186828,


AI626081, AI275985,


AI494518, AA749446,


AW089880, AI914010,


AW207720, AI130746,


AA976621, AI921928,


AA993271, AI130728,


AI299980, 819745,
884393,


W69667, AA742981,


AL046248, AA229014,


AI279330, AA156866,


AI216523, AA255782,


AI811823, AA488821,


AI419263, AA489068,


AA229863, AA705638,


AA701250, AA229731,


N47318, AW271763,


AA687125, W73367,


AL044304, AA862901,


AW272381, AW372345,


AA939081, AW021871,


W73428, AI478491,


AA702739, AA977247,


AA765337, W45513,


AI141957, F06156,


AA127066, 884392,


AI283582, AI985183,


AW439593, AI186368,


AI687916, AI560741,


AI203537, AA047572,


W69666, H66133,


AI081094, AA453238,


AA356896, AA463404,


AW294792, AA318684,


225224, H66549,


AA373158, AA011074,


N92391, AA480226,


AW292636, H43300,


AI222506, AA378963,


AA492470, AA579885,


T47922, W24708, T47923,


234


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA348753, T05906,


AA455850, H43299,


AI419633, AW292633,


AA011075, D61430,


AI587496, AL133683,


AA147480, AA772416,


AA325615, 845164,


AA714016, AA130104,


AI560507, AW272813,


AW376847, AI471022,


H16172, AI568064,


AA378964, 868334,


AI567645, AA256004,


AA341472, AW376809,


W45654, AI401496,


AA280280, AI905674,


AA887264, AA147880,


AA704633, AA429202,


AI905673, AI216605,


AW 168495, C 14179,


AI963999, AC020663,


U70255, AC002094,


L06564, U72484, X89268,


and AF005380.


HTLDS55 134 1182304 1 - 1302 15 - 1316AI890919, AI018797,


AA913452, AI797580,


AI809012, AI187382,


AA448485, AI554914,


AW137847, AI393577,


AA382830, AA432050,


AA609003, and AC020663.


HWMAE53 135 1187258 1 - 436 15 - 450 AA368584, and AB023227.


HTODG16 136 1057155 1 - 591 15 - 605 AW403969, AA252781,


and AL041242.


HFXCG28 137 1199587 1 - 1454 15 - 1468AL080117, AB023176,
and


U14103.


HFTCU45 138 910053 1 - 538 15 - 552 U47343, AA325176,


AA463333, and AA323357.


HFTBL33 139 1222661 1 - 2136 15 - 2150AI677902, AI338780,


AI684570, AI928887,


AW136644, AW264299,


AI652923, AI089627,


AI298483, AI372875,


AA463846, AI141311,


AW129570, AW204313,


AI374899, AW 134722,


AW305132, AI366527,


AA463333, AA031465,


U47343, AI654427,


235


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW205086, W26816,


AI217285, D81166,


AA906309, AI382756,


AA031486, D80712,


AA054522, AA325176,


AA884159, AA323357,


AA247154, N71729,


C15737, N71226, AI557264,


AI541393, AI557278,
and


A84916.


HCEPH84 140 1148128 1 - 1499 15 - 1513AA442393, AA019932,


W76203, AI372800,


856176, AI372801,


AW248425, AA350199,


T77421, AA054057,


AI298004, T75382, T47787,


AA018176, F08410,


AA058754, 855903,


836279, AA191072,


AA180848, AA194490,


F13127, AA193053,


AA326216, 816315,


AA663485, AA166864,


AA204736, AA249839,


F05591, AI651722,


AA167722, 242609,


AW 138074, AA339074,


AA054202, AA056327,


AI479964, AA207119,


AI221332, F26963, F36674,


F28731, AI937722, F36659,


AI609568, F25641, F25571,


T33292, AA058465,


F33105, AA206829,


AA436615, 849566,


855818, AA193004,


AI194037, 856064,


AA019920, AA191589,


851792, T51483, 225123,


F23450, AA054088,


838363, W72966, 241401,


AA054025, F01857,


AA194690, F10729,


F17797, F35038, F02117,


263803, 264258, 263848,


261697, 261696, and


264257.


HHFON19 141 1061675 1 - 333 15 - 347 AA356476, and AC003072.


HEMCL65 142 910900 1 - 453 15 - 467 AI902552, and AI902562.


236


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
HMAMB94 143 910909 1 - 627 15 - 641 AA158332, 890961,


890977, and 890972.


HFICR59 144 1171979 1 - 1021 15 - 1035 AI190165, AI979249,


AI917302, AI633819,


AI624750, AI471728,


AW196791, AI985423,


AI471611, AA609421,


AA705338, N22327,


AA811162, N75202,


AI922484, H79904,
H79810,


AW407702, AF162130,


AC005084, and AF161181.


HSXDD55 145 911460 1 - 1164 15 - 1178 AA446069, H19388,


AA121710, AA429913,


F07861, H12126,


AW104301, H55229,
and


AB002349.


HCQCI06 146 915000 1 - 1190 15 - 1204 AI123952, AI751915,


AA343597, W79144,


AA345718, AA304549,


AA256582, W81545,


D45547, AL117664,


AC068763, AC068763,
and


AC069223.


HPMLJ44 147 1199601 1 - 1771 15 - 1785 AA778721, AI309326,


AI923088, AW157189,


AW009559, N21676,


AW277241, AA056157,


AI015567, AA142926,


AA935517, N71214,


AI340068, AA046446,


AI890415, AI760248,


AI421490, AI423473,


AA576688, AI342399,


AA405935, AA946956,


AW403205, AA826534,


AA143149, AA313266,


AI803454, AA594414,


AA233629, AA125818,


N22011, AI862039,


AA315935, AW 148924,


AI245757, AW374039,


N80237, AA488928,


W33046, AW304989,


AI953431, AA782164,


H89121, T78059, AI344475,


AA074821, AI148049,


AW163161, AI707964,


AA702036, AA864308,


237


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA034997, AAOS8701,


AA04S662, AA172038,


AW0576S1, AA084S70,


AW438849, AI708S66,


AA743021, AA13S017,


COSOS3, AA04S663,


AA3120S9, AW268698,


AW078896, AA047840,


AA894905, AA724345,


AAS863SS, AA947891,


AAOS3980, N31083,


AI024387, AA878972,


N31289, AI631228,


W38328, AL048109,


F13274, TS9457,


AA172290, AA831417,


AI90S071, AA12S949,


H06S42, AA018173,


AW337421, H06484,


T06616, AA8370S9,


T77300, AW295S81,


AA7S9329, 839702,


Z3993S, F10872,


AAOS7237, T36230,


H89228, AA373169,


AA233777, AAS98442,


DS3439, AA03S4S9,


AAOS6096, AA484066,


T19033, 896192,


AA693386, AL048108,


AW392317, AI090106,


AA732389, AW 150491,


243869, R3818S, AI270737,


W04S96, 237004, T18978,


AAS01472, AA483236,


AA383381, H41069,


AA749100, AI200780,


AA079S74, AAS80SS3,


AA40S183, AA079S26,


AW189389, NS6489,


D19937, AI90S101,


AR044461, and AF061739.


HNTCUS 148 12234791 - 2837 1 S - 28S AW390189, AW068631,
1 1


AW068632, AW083013,


AW299875, AA042847,


AA868916, AI632320,


AA861695, AI823835,


AL039139, AW340822,


AI700370, AA97S794,


238


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI342192, AI928261,


AW390370, AA480338,


W78034, N36497,


AI635086, N66488,


W56029, N41511, H42127,


AI307661, AI610367,


AI735104, AA643969,


W40245, AI087138,


AI383572, W40506,


AI570860, AI089368,


AI375757, AI813851,


H42126, AI248569,


AI243286, H03624, N25781,


N25786, AW291887,


881326, W07239, N36492,


AA243297, AI221570,


AI140829, 881575,


AA101761, AW195338,


AW390411, AI888835,


AA463566, AI206788,


N78809, AW074503,


H03625, AA778483,


AL041056, N52929,


AL039152, AW452298,


D79350, AW392781,


AA514883, N40562,


AI583048, AA249501,


AI125441, AA382265,


D79601, N46605,


AA248652, AA044396,


AA299610, AI221697,


D25811, AA405866,


AI932292, and AL049934.


HNFD052 149 916260 1 - 345 15 - 359 AA357035, U23853,


L11329, and AC012307.


HSHBW40 150 1144361 1 - 1120 15 - 1134AA393046, AA449274,


AI417305, H54361,


AA450152, H89797,


AA045551, AA315053,


W28083, AI925931,


AI810820, AA310873,


883856, 833893, T78727,


818673, AA306399,


AA306395, AA126773,


AA324948, AA450094,


AW379133, T77732,


T27901, W25779, T51655,


W25559, AW189057,


814561, T78180, H48688,


239


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW365287, AI342499,


W25778, 856439,


AI858990, AA374368,


AW362215, AI609827,


AA344165, AI655305,


AA121371, H77659,


818418, 834534, H87657,


AA378311, AI269259,


808800, AA366795,


N99789, AW365290,


N51168, 855512,


AW378738, AI903043,


AA609451, AW378748,


AA493146, AA480655,


AI874365, 834545,


AA862146, AA931827,


AA864423, AW450406,


AI128080, AW378763,


887869, AI139593,


AA716045, AA857294,


AI093259, AA665796,


AA890188, AA828411,


AI271946, AA255578,


AA121361, AA628775,


AA126760, AI343939,


AI373574, AI197888,


AI261530, AW003083,


AAl 14177, AI744469,


AA449220, AI339832,


AI337024, AI885656,


AA311426, N92854,


AI870955, AI680690,


898212, 898213, 856440,


AI935195, F06274,


AF225971, M61764,


AB015946, and AC004985.


HHEJR23 151 12006431 - 930 15 - 944 AI440338, AA868192,


AI808409, AI567414,


AI278014, AI291975,


AW304837, AW024447,


AI341786, AI183586,


AW024630, AI208559,


AI302861, AA043196,


AI492200, AW 103254,


AA731505, AA741444,


AI160734, AI868969,


AI214841, AI469608,


AA947737, AI351129,


AA045490, AA043598,


240


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA970703, AI095776,


D25707, T19154, AI187765,


AA058909, AW087623,


AA652268, AA507020,


AA291791, AW449034,


AA434511, AI809374,


AW087534, AI571861,


AI273142, AI247193,


AI886206, AI280747,


AI681985, AL120736,


AI621209, AI611348,


AI817552, AI932794,


AW 149227, AI817543,


AW084786, AW088793,


AW302988, AI564247,


AI554821, AI270183,


AI637584, AI925156,


AW075413, AI420521,


AI497733, AI567351,


AI524671, AW071417,


AI318280, AI955906,


AI680498, AW081036,


AI634224, AI572787,


AI174394, AW083175,


AW 129106, AI802542,


AI978703, AI499263,


AI335426, AI348777,


AI539808, AW188382,


AI864836, AW085906,


AI567612, AI648684,


AI783792, AI499146,


AI554485, AI628324,


AL042628, AW074172,


AI473554, AI493576,


AW198090, AI800453,


AI500077, AI871923,


AI918655, AI829327,


AI571439, AI620639,


AI572418, AI929108,


AI800433, AI280661,


AL134999, AI620015,


AI345416, AI345612,


AI869367, AI624693,


AI889376, AI917252,


AI241819, AW082033,


AI570781, AI536685,


AI345415, AL040243,


AL036146, AI252023,


AW302992, AI280751,


241


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI866770, AI824648,


r
AW083750, AI926790,


AW020693, AW169671,


AI538716, AI683475,


AA225339, AI619607,


AI439717, AI365256,


AI476109, AI560099,


AI492540, AI537303,


AW080992, AI818980,


AL046942, AW023590,


AW151786, AW130776,


AW152469, AL038605,


ALl 19863, AI285735,


AI758437, AW079654,


AI312542, AW 131282,


AI383919, AW090700,


AI873644, AW118382,


AI564719, AI269205,


AL042551, AI582932,


AI282504, AI828705,


AI590118, AI288285,


AI801322, AI801544,


AI269862, AI698391,


AW 167410, AI269696,


. AI784252, AI344928,


AW074869, AW 166865,


AW054931, AI431909,


AI619502, AI862139,


AI569583, AI440426,


AI306705, AI670002,


AW 169653, AI612759,


AW 152186, AI591420,


AL121014, AI636456,


AW105601, AI537617,


AI445165, AI680435,


AI621179, AI619716,


AI696606, AI866090,


AI476046, AI924686,


AI886753, AI923370,


AI572892, AI866798,


AI916419, AI818683,


AI431424, AI613270,


' AI242931, AI886124,


AI591228, AI624548,


AI684036, AI961286,


AL042382, AL036980,


AW131331, AI826225,


AI963216, AI433157,


AW088899, AA508692,


242


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW303075, AL038445,


AI612885, AW080746,


AA012905, AI571909,


AI564426, AI863382,


AI271786, AL043326,


AL036396, AI608936,


~' ,..~, ';A..W268122, AW059837,'
'


AL134830, AI537677,


AW089572, AW132001,


AI349957, AI815855,


AI537991, AW080402,


AW 190042, AI922676,


AW103371, AW073994,


246372, AL137459,


AF 113690, I89947,
I48979,


AL117585, AF118094,


I48978, AL049314,


AL117460, AF177401,


A08916, I17767, A08913,


S68736, A08910,


AF118070, AL137271,


E02349, A08909, U42766,


AL137557, AL117435,


X96540, AL122098, I89931,


. ' _ . _. . , I4962~5,, AF158248, _.
' .. . .


.. _ , . . .-~- .x'104032, AF183393, , ~=~':
_ .


X65873, AF113694,


X72889, AL110221,


AF090900, AL137463,


Y16645, X82434,


AL080127, AL122123,


AL122093, AF113019,


AL050024, I33392,


AF111851, X93495,


A03736, U00763,


AF017437, AL137550,


AJ238278, AL133075,


AF090903, AF125948,


AL137527, AL050393,


AF079765, AF067728,


AL133640, AF078844,


AL049452, A77033,


' AL049464, - . -
' - - -:A~77035 '


w~~'' . ,
vY11587, AL133557,


AL110225, X63574,


AF118064, U67958,


AF113699, 578214, 282022,


AF113676, AB019565,


A58524, A58523,


243


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF090934, AL110196,


AL049382, AL137648,


X84990, AL133016,


A12297, U72620,


AL137283, AF090943,


AJ242859, E07108,


AL050146, Y14314,


AL133606, AF106862,


AL049466, L31396,


AL080124, U80742,


AL137521, L31397,


AL080060, A65341,


X70685, AF017152,


AF146568, AL133565,


I03321, AF091084,


AL049300, AL133093,


AJ000937, AL122050,


AR059958, AL117457,


AL133560, AF026816,


AL080137, U35846,


AL122121, AL122110,


AL133080, AL049430,


AL050277, AL117583,


AF113691, AF113013,


AL050149, AL050116,


AL050108, AF090896,


AF090901, AF113677,


AF097996, Y11254,


AF125949, AL133568,


AL117394, AF061943,


AF113689, AL050138,


E03348, AL080159,


AL137538, AR011880,


AL133113, X98834,


A93016, AL049938,


AF087943, E07361,


AJ012755, AF119337,


A08912, E15569,


AL096744, I42402,


AL110197, U91329,
I09360,


AL137560, I26207,


AL133072, AL049283,


AF026124, A93350,


AL133077, 561953,


AL122049, AL137556,


AL133014, AF081197,


E08263, E08264,


AL133104, AR000496,


U39656, AF111112,


244


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AL110280, AF003737,


I00734, Y09972, E00617,


E00717, E00778,


AL050172, AL137476,


AL137480, AL137523,


U96683, AR038969,


AF153205, Y07905,


AF061573, AF079763,


A07647, AF185576,


AF081195, U68387,


AL133067, 272491,


M30514, AF162270,


A45787, E05822,


AL080074, E08631,


AL117440, AJ006417,


AR038854, A90832,


AL117416, AL133098,


AL137294, AF057300,


AF057299, E04233,


U78525, AL137526,


U58996, X92070,


AF106827, L30117,
237987,,


U49908, U35146, L19437,


X87582, AL137292,


AF210052, AL137533,


AF111849, AL137529,


AF008439, AF067790,


AR013797, AL133665,


E02221, AF032666,


X62580, AL122111,


AL133031, AF051325,


AL117432, I41145,


AC002467, AL137478,


AF132676, AF061836,


AL122118, AL137488,
and


AL050092.


HCFMW71 152 1199561 1 - 1439 15 - 1453 AI923217, AA197141,


AI814569, AI762903,


AI804682, AI804663,


AI143304, AA197116,


AI082030, AI911904,


AI139520, D53214,
892413,


AW274978, W93272,


AI183410, AW450838,


AI216343, AA680119,


AI918971, C15320,


W93273, F00607,


AI823928, T51116,
219397,


T96093, 892414, T51024,


245


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI422647, AI216344,
AI741425, AI936703,
817362, T96094, 842713,
AI869077, F00114, C00216,
266165, 256024, and
AW662096.


HBODA38 153 923456 1 - 1882 15 - 1896AA196401, W79841,


AA112831, AA197134,


C05212, W 19681,


AA195812, 225012,


AI004215, 230134,


AA194641, AI809925,


AA258550, 228480,


w F29133, AA699576,


F20387, AI129717,


AA194359, AI079487,


AA195355, AA195094,


AA931319, F34387,


219343, F36761, H67895,


AA258604, AI378821,


F19321, AI089231, 891150,


AI702453, AI288969,


AI313118, AI351843,


F31038, N56278, AI381419,


AA195356, AA131264,


AA195051, AA192095,


AI264911, and AA086284.


HCYBK19 154 925494 1 - 2664 15 - 2678AI829726, AI809562,


AI927757, AI927758,


AI927768, AW297770,


AA165336, AI638072,


AI149852, AW298053,


AI365991, AA904547,


H15885, AA985065,


AI359235, AW207262,


AI470080, AI421572,


AA305419, AA985170,


AW341104, AI423550,


AI362528, AI453702,


AI654827, AA927292,


H91991, AI365149,


AI365324, D81295,


AI797740, AI357382,


AI953516, AA909430,


H01576, AA969956,


AA379840, H52748,


837302, AA905264,


243289, 862753, 818887,


868272, H04859,


246


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA746048, T31617,


H04764, T78758, 244077,


T32807, D60323,


AA634475, H01477,


AI193962, H00918, D81446,


M86147, AA983881,


AA318875, N64328,


AA165369, 239361,


D62629, T16370, F03272,


H00919, N34301, and


H46107.


HOFNH30 155 928365 1 - 362 15 - 376 AF186380, and AF127138.


HFXED03 156 1136469 1 - 1594 15 - 1608AI640298, AI984640,


AI927459, AI492589,


AA037170, AA985228,


859151, M86008,


AI539361, AA480185,


W25874, AW070463,


T35581, AA354520,


220600, AA853067,


AA151003, AA374936,


AA374649, AI693470,


W81208, AI765122,


AI970100, AA332604,


AI129029, AI373608,


F06971, F06054,


AA446917, H04913,


AA853066, AR035972,


AL110300, and A75455.


HWMEV63 157 931154 1 - 440 15 - 454 D13626, and AC078816.


HWHGT26 158 1147962 1 - 2634 15 - 2648AA829643, D82406,


AW339052, 236241,


AA885340, H66744,


AW440614, H85794,


H96697, AW 105208,


H12642, AA282102,


225297, AA281700,


AA292399, H86205,


N58359, AA548845,


H72231, AA938721,


H58538, AA333403,


H12593, AA568870,


AA281638, 228896,


AA282103, H86563,


AA418431, H84902,


AI685214, H71667,


AW075858, AA298135,


H66743, AA653194,


L34968, AF065392,


247


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF013966, AF065391,


AF013965, AF013967,
and


AF133818.


HBXBG65 159 932780 1 - 521 15 - 535 836281, AF094480,
and


AF094479.


HSDHB12 160 941973 1 - 624 15 - 638 AA447298.


HLWAR77 161 947484 1 - 1275 15 - 1289 AA449919, AA449920,
and


AF119815.


HTTJQ27 162 1182680 1 - 1043 15 - 1057 AA522440, AI683658,


AA527385, AW068809,


AI567764, AI962065,


H61360, AI962103,


AA811386, AW 188595,


AW 194232, AI953393,


AI868180, AI470299,


AI241678, AW074057,
and


AL050280.


HUJDA09 163 1153887 1 - 767 15 - 781 AI133670, W26633,


AW375605, AA318069,


AA853744, AI174232,


AA521255, AA024888,


N46555, 858116,


AA447471, H77993,


858328, T47555, AI205304,


T81235, W25885, 833242,


AA349799, 220968,


AA206898, AW380305,


AA852419, AA355036,


AW352357, AA486937,


T80568, AA350303,


AW138906, AA533322,


W22449, AI372724,


AI751908, AL133628,


AF 124440, AJ 13303
8, and


AB029448.


HEOST94 164 954582 1 - 634 15 - 648 AA243527, AL133847,


AW070995, 850115,


H17151, H11956,


AW245691, W81176,


AA324883, AA134693,


AA322031, 243085,


AI908431, H10325,


AB007857, AC021705,


AC021705, and AC006435.


HUVHQ75 165 955032 1 - 620 15 - 634 M86008, T35581, 220600,


AI693470, AL110300,


AR035972, and A75455.


HWAGC08 166 1189787 1 - 1417 15 - 1431 AA133439, AI150395,


AI394694, AA836507,


248


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI904808, AA133438,
AA055801, AI216678,
AA482998, AA055852,
AA447236, AL048981,
and
AC005071.


HMWJI52 167 1202746 1 - 271315 - 2727 AI659483, AI857996,


AI745593, N63916,


AA541651, AI85801
l,


N63903, AA573858,


AW149456, AW150971,


AW172771, N53446,


AI568454, AA857892,


AA532378, AI185005,


AI040894, AI740569,


AA876133, AA677515,


AI057543, AA865457,


AI151432, AI095739,


AA425718, AL118870,


AW188153, AI383493,


AA879134, AI093353,


AI970686, AA973591,


AA918313, AA708914,


AW 129493, AI271887,


AI016698, AI093351,


AW363695, AA682557,


AA586346, AA115282,


AW 189896, AA426545,


AA738302, AI139340,


AA834075, AW363660,


AA158540, AA291139,


AI299505, AI672698,


AI025591, AA705475,


AAl 15260, AI360357,


AI927241, AI348355,


N98443, AI537223,
N98274,


AA741220, AA835860,


AA291762, N25158,


AA460612, W69122,


D81559, H63158, AI246009,


H15065, T17447, 826864,


AA974875, D52505,


N95190, AI271787,


AA721273, H16393,


AW149360, AA135600,


AI245518, AA135540,


AA806170, AI187006,


AA316474, T15919,


AI272340, AA290769,


H08014, H54356, 238773,


249


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
894384, 850814, H63074,


H59564, H14744,


AA213527, AA724344,


AA743446, H84049,


AA917576, H19010,


N92062, H19306, H07918,


D61231, H54277,


AA767619, AW 118549,


AA766172, N68849,


T30190, AA548822,


AA533504, F08853,


AA334162, AI203502,


F05055, F04359, AI204122,


AI537924, 242850, H84050,


N55132, H13528,


AA564728, H05319,


F11186, F02319, AI368540,


H15064, D51046, F04602,


826718, H06692, F01594,


894795, AA486318,


844372, 827956, 847421,


827093, H83828,


AA740665, AA354950,


AA917561, H59565,


AA995419, 814815,


D19736, AI302335, 843854,


894714, 826942, 840121,


H06693, 824567, 242594,


AA705980, AA342628,


N72449, 827965,


AW295138, 242064,


AI090231, F08378,


AI381196, T27000, D56273,


AA486216, 835155,


AA214543, W24591,


D59997, AA059019,


AA948086, 278361,


T27001, AA158539,


H05373, H86329, 245494,


AA938906, AA702132,


AA300225, 257544,


259851, 259832, 259966,


H98637, AW470568,


AW513873, AW591435,


and AW769993.


HAABH11 168 1199572 1 - 851 15 - 865 AI418830, AA424650,


N25267, AW026561,


AI126772, H83701, N22732,


AA631750, AA418447,


250


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA759336, AI250627,


r
AI251221, AI494201,


AI521005, AI472536,


AI540606, AI633300,


AI401697, AI687568,


AI633061, AW059713,


AL046933, AI446373,


N29277, AI261589,


AW 167157, AI799540,


AW 162194, AA903067,


AL038605, AI537837,


AI446515, AI702527,


AI916419, AW 168503,


AA948318, AI804505,


AI491904, AI468959,


AI573026, AI933992,


AI432570, AW024921,


AI267185, AI537643,


N25033, AI284060,


AI345688, AI678875,


AI453328, AI377001,


AW081383, AI340726,


AL119511, AI560545,


AW411359, AI434731;


AI583578, AW 161202,


H89138, AW081186,


AI560679, AI636619,


AI696583, AI628325,


AI584118, AI288843,


AI590423, AI679550,


AA848053, AI690706,


AI702065, AW 193467,


T49776, AI810544,


AI582871, AI623736,


AI567540, AI345567,


AI890533, AW081234,


AI282031, AI659795,


AI698401, AI445588,


AI349957, AW 172723,


AI242736, AI285514,


AI274614, AA729017,


AI345005, AI636788,


AL040586, AW152182,


AA088789, AI802372,


AI828734, AI336633,


AI357599, AW022682,


AI866461, AI559863,


AI344931, AI338060,


AI559752, AW262565,


251


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW366372, AI921092,


AW148589, AI539800,


AL046942, AA503384,


AI656270, AI815915,


AI287449, AI250646,


T99953, AI247306,


AI952914, AI373622,


AI471282, AL048644,


AW082532, AI922110,


N29481, AI811603,


AI890051, AI355277,


AI310709, AW 196299,


AI814218, AI538850,


AI634251, AI819663,


AW021373, AI624120,


AI284131, AI860652,


AI829330, AI282326,


AW022636, AI263584,


AW080076, AW083572,


AI824748, AW079315,


AI671651, AI365256,


AW410850, AI932458,


AI554444, AI050666,


AA587590, AI829327,


AA502794, AW 148882,


AA070889, AI961403,


AI690536, AI270039,


AW162189, AI932739,


AW 189148, AI872423,


AI598209, AI537677,


N95566, AI955866,


AW 151926, AI345143,


AI479030, AI434038,


AW409801, AL119399,


AW083168, AI620864,


AI868204, AI584130,


AL048323, AI765323,


AI886022, AI612885,


AI434242, AI799189,


AI919107, AI571699,


AW265004, AI636507,


AL048340, AW 104062,


AW 148970, AI962040,


AW237096, AI473451,


AI307505, AI582932,


AA580663, AI570056,


AI161016, AW263804,


AW 194014, AI690813,


AI370392, AI281867,


252


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW191844, AI888621,


AI691088, AI274655,


AI469516, AW079334,


AA808175, AI287476,


AW 161098, AI949968,


AI345014, AI589428,


AI355017, AI926333,


AA575874, AI559524,


AL049053, AL040449,


AI281659, AI203903,


AW196075, AI884459,


AI440238, AI345224,


AI678446, AA928539,


N98606, AI915210,


AW 103923, AI687809,


AW129731, AI345253,


AI096534, AI289337,


AI283143, AI469674,


AI818320, AI589218,


E12579, I48978, AF146568,


AL050138, I89947,
A08913,


AF098162, A08912,


A08910, A08911, A18777,


A08909, AF113689,


AL080154, A08907,


536676, AL109672,


AF067728, A08908,


AF111851, AF153205,


576508, AF119337,


U77594, U49434, L13297,


S77771, I89931, AF113013,


I49625, AR038854,


AL137547, I89934,


AL137271, U57715,


Y13350, AL133062,


AF047716, AR068466,


E12580, AF112208,


AF079765, AL137665,


AF115410, 282022,


AF183393, U57352,


AF081571, AL0801,37,


AL133619, AL133072,


U49908, AL133623,


AF113019, AF017152,


E15582, AL110221,


A08916, A15345,


AF085809, 554890,


AL 117648, AF 114170,


I33391, AL049382,
I42402,


253


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF143957, AF114818,


AL080127, X57084,
J05277,


AF104032, I89944,
I48979,


AF1622'70, Y11587,


D89079, AF094480,


AF158248, X67813,


AL050092, AF061573,


Y08769, E02914, 561953,


L04849, AF003737,
L04852,


AR009628, AF067420,


A45787, AR068182,


AF182215, Y14314,


AL080124, A32826,


A30330, A32827, A30331,


X98066, AF012536,


AF036941, X99257,


AL133070, AL049460,


X52128, AL122045,


AL137705, AL0800'74,


X93495, AF113691,


AF167995, A57389,


AF100931, AL137526,


AL133093, U92068,


U76419, A83556,


AF118092, AL122111,


AB025103, AF159615,


AL137554, J05032,
E08631,


AF036268, AL080140,


AF015958, 568736,


AL137530, X66975,


AL050393, AL137555,


AL110222, E01614,


E13364, X62773,


AL050366, E15569,


AC004227, 578214,


AC004822, AL137539,


U75932, 583456, AJ001838,


AF090934, Y 16645,


X79812, AL110196,


AL137640, X83544,


AF118090, AL080110,


AF017790, AL117649,


AF047443, U37359,


AF113676, AL137533,


AF039138, AF039137,


AF044323, AL137488,


S79832, E06743, X65873,


AL133049, AF022363,


X99226, X53587,


254


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AC002382, I29004, X66417,


AF077051, AF078844,


575997, AL133054, L10353,


AL049464, A07588,


U89906, AL050277,


A77033, A77035,


AL117587, U58996,


X89102, D00174,


AF111849, AL117460,


AF126488, AL080139,


E01314, U96683,


AF058921, A08915,


AL137256, AL137558,


I66342, AL117440,


AJ010277, AL137658,


AL133014, U80742,


AL080126, E02221,


A21101, M80340,


AC004200, E02152,


AF106945, AF169154,


AF013214, AL122049,


AR068753, X56039,


A41575, A65341, I18355,


AF065135, A76337,


AL133640, I34392,


AF076633, AF200464,


AJ242859, AR038969,


A27171, AF004162,


Y14634, I03321, A12297,


AJ012582, AL137641,


U36585, AL137292,


U89295, I68732, A17115,


A18079, X72387, 249258,


AL137463, X97332, I41145,


AF118064, I09360, and


AF 118070.


HAPAI15 169 1187435 1 - 161715 - 1631 AW402614, AA662103,


AI929056, AW409948,


AA311813, AA310195,


AI816311, AW294565,


AA976304, AA731168,


AA251463, AA205378,


AA805414, AA053460,


AW401973, AA312734,


W23120, AA195341,


AA352713, AA195340,


AA662069, AA317160,


AA354224, T85806,


AI762990, AA297895,


255


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA35661 l, AA315788,
AA281670, AA283373,
T93968, 221401, D53904,
D53947, T89686, T89509,
AA053212, AA448152,
D50918, AB023622, and
AC004913.


HNGBQ66 170 1172819 1 - 1046 15 - 1060AW239349, AA375315,


. AW 151247, AA355780,


AI452836, AL036896,


AI801563, AA584241,


AA493546, AI620354,


AI360521, AW275432,


AW 117860, AL041375,


AA630476, N92588,


823873, AI224583,


AW272389, AI473671,


AI797998, N67313,


AL044701, AI308529,


T03928, AI889995,


AI049999, AI267285,


AI355246, AI734193,


AA455202, AW327852,


AA666295, AA676592,


AI653776, AW264548,


AI761677, AA809125,


AW021674, AI288033,


AA526508, H71678,


AA507623, AW 177869,


AI732682, AW072963,


AW082104, AI734158,


H60912, AA814503,


AA629759, AA493789,


H38769, AI798041,


AA584207, AW117704,


AA526413, AW239465,


AW008184, AI188049,


AI336771, AA558488,


AW301483, AA199578,


AI921706, AA594742,


AI567676, AI872229,


AI090377, AI310670,


AA629713, AL036949,


AI590404, AA533660,


AI369076, AA658443,


AA846923, AA226356,


F35684, AI174703,


AI791426, AI732473,


AI283938, AA225392,


256


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI469599, AI349130,


AI031759, AA804726,


AI924950, AI683079,


AI613459, AI309943,


AA457592, AL117554,


AC005049, AF205588,


AC005215, AL035659,


AC003663, AL035413,


AC004099, AL096791,


AC006544, AF126403,


AC005500, AC005516,


AC007066, AC004913,


AB001523, AC005730,


AL035086, AP001052,


AC007934, AC002310,


AL080243, AF207550,


AL034420, AC002544,


AC004491, AL021391,


295118, AC005391,


AC007685, 282179,


AC005746, AF064861,


AC018633, AC002316,


AP000501, AC005529,


AL022326, AL022318,


AC005602, AF109907,


U65896, AL031848,


AC004859, AC005944,


AL031427, AC007955,


AC004755, AC005913,


AC005921, M89651,


AF038458, AL049856,


AC005088, AC005535,


AB023048, AC004675,


AC004816, AL009172,


AC005933, AC000115,


AC005899, 284466,


AC007226, AF088219,


297632, AC004797,


299716, L47234,


AC006059, AP000503,


AC005578, AF030453,


AC005231, AL021453,


AC002365, AC004263,


AF023268, AB000882,


AC005914, AC004149,


AC005081, AL035455,


AC006236, AL031255,


AC006011, AC005527,


AC005544, 295116,


257


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AL133448, AL031587,


AC004973, AL096701,


AL136295, L78810,


AP000045, AL031282,


U89335, AC004000,


299128, AC006539,


AL049697, AC007308,


AC001231, AC019014,


AC004882, AL049646,


AC007382, AC004583,


AP000356, U96409,


AC007021, 299291,


AC005069, AC006479,


AC003030, AC004686,


AP000300, AC006084,


AC005253, L78833,


AC005553, AJ246003,


AC006211, 284480,


AP000228, AC007773,


AC004655, U91328,


AC004673, AL022311,


AL109801, AC004124,


AC005523, AC002119,


AF060568, AP000279,


AF134726, AL035461,


AC006468, AL035423,


AC004820, AJ003147,


AL121603, AC005412,


AC003665, 268284,


AC004804, AL021398,


AL024498, AC005670,


AC007298, AL022316,


AL035587, Y18000,


AL109963, AL031678,


U91318, AB000931,


AC005186, AC005325,


AC005828, AL139054,


AC005763, AC002350,


AC004876, AC005666,


AL049569, AC006026,


AC002477, 297056,


AP000140, AC004812,


AC002400, AC005067,


AC005512, AC000394,


285986, AC004878,


AC005874, AF134471,


AC004692, AL049694,


AP000113, AC004638,


AL033392, 269721,


258


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AC007687, AC006480,


AC007731, AL031281,


AP000106, AP000038,


AC004771, AC005300,


AC004552, AC006162,


283840, AC005082,


AC005587, AC006077,


U47924, AC010205,


AL050312, AC006039,


AC005379, AL008582,


AP000512, AC006538,


AL121652, AP000694,


AP000692, 295115,


AC005005, AC006205,


AP000689, AC004477,


298743, AC004858,


AJ229041, AL021806,


AF015416, AC002395,


AC005280, AL035405,


AC002558, AC005071,


AC005207, AL034417,


AL049540, AP000359,


AP000039, AD000812,


283844, AR036572,


AC006536, 293017,


AL021917, AC007390,


AC007263, AP000280,


AC007041, AC004031,


AC020663, AC006581,


AL031133, AF011889,
and


AC004167.


HTXPY09 171 981988 1 - 1145 15 - 1159 AI435592, U56656,


AA148805, AA046175,


AA075198, AF194371,


AF069782, AF053232,


AF161469, AL117554,
and


AF123534.


HCFLJ17 172 1193629 1 - 1769 15 - 1783 AW148724, AW236350,


AW058251, AW363242,


C75194, AA773368,


AA152132, AA31~3387,


AA047345, AL121232,


W38779, T39704,


AA404974, AA377300,


AA047344, AI050068,


AI633499, AW363273,


AA362228, AA121624,


AA247511, AW029375,


T40740, AW392857,


259


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
W07426, D51246,
AA173649, AF155096,
AF135439, and AW769129.


HGOCA12 173 1172054 1 - 1234 15 - 1248AW444890, AA461185,


AA435513, AA602372,


AA723271, AA774585,


AI971328, AI004443,


W72923, F36785, F17742,


F19634, W94217,


AA346094, AA346095,


F24441, AA393548,


AA461361, and AB027004.


HCE5E94 174 1070802 1 - 1465 15 - 1479AI142098, AI061467,


AA555087, AI816028,


AI064696, AI132975,


AI955216, AI954923,


AI185092, AI961492,


AA557261, AA594123,


AI683136, AI821060,


AA167739, AI492571,


AA167833, AI128382,


AI039982, AI801532,


AI884567, AI708278,


AA877359, AA771931,


AI225219, AW 169567,


AW149057, W02976,


AA570678, AA847293,


AA738472, AI916354,


AI568477, AI687057,


AA570139, AA485692,


AI075416, AW025307,


AA009525, AI870831,


N79600, AA524522,


AA128450, AA447933,


AA084924, AI733764,


W 19067, AI630317,


AA972196, AA037503,


AA113991, AA614719,


AA112786, AI191141,


AI815873, W28377,


AA846646, AA037371,


AA165667, 836946,


AA583651, AL134404,


AI354274, AI436348,


AA729500, AW 169930,


AI288012, H08475, F28921,


AA224339, H09582,


N72263, 240082, M79262,


AA854626, AI582426,


260


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AA485823, AA857288,


AI811622, T53473, 851376,


AA085027, AA165631,


C20581, H67335, T53474,


F04102, AA009524,


N28590, H53206,


AA086264, AA322093,


H08757, AI868288, F37189,


843209, 811825,


AA113990, AA372866,


AI203752, N67107, H09038,


F04097, 817117, N87606,


AA365003, 895092,


AA088569, AW027974,


AA764799, AI474701,


AA170849, AA224133,


AA886226, AA366031,


AI572914, AA903493,


AA748748, AF087661,


AA076638, AA916592,


AI088936, AI089690,


AA447640, AA448044,


AI014970, AI362134,


AI432519, AI471047,


AI681970, AI969201,


AW475066, and


AW517258.


HNTAV78 175 971315 1 - 528 15 - 542


HHEEL28 176 1216492 1 - 1918 15 - 1932AW138424, AA831257,


AI801844, AI028474,


AI692891, AA767936,


AW188936, AI648511,


AA359127, AI970093,


AI381789, AI364659,


AI697316, AA358752,


AA320236, AW440942,


AI819663, AL079799,


AI264299, AI685087,


N29277, AL134832,


AI963625, AI539260,


AI349012, AI401697,


AW 188525, AW020397,


AW020561, AL039011,


AI333104, AW129148,


AW167918, AI336575,


AI687568, AW051088,


AW080076, AW 148970,


AI250627, AI539690,


AL119399, AI434731,


261


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AL036954, AI433611,


r
AI538055, AL037454,


N75779, AW020328,


AW301344, AI884318,


AI690620, AI318603,


AW 170725, AA808175,


AI419826, AI890576,


AW303152, AI537643,


AL119511, AI559558,


AI538805, AI345778,


AI440294, AW028416,


AI889372, AA903106,


AW023859, AW195253,


AW 162315, AW 163554,


AI500061, AI925685,


AA830396, AI890223,


AI368579, AI560545,


AI680369, AW080290,


AW024793, AA761557,


AI561356, AA737649,


AI540606, AW021662,


AI469516, AI050881,


AI284060, AI374987,


AW074057, AA693331,


AI554245, AI537677,


AI538028, AW020592,


AI225000, AI621341,


AL038076, AW160376,


AI698391, AW022494,


AL041331, AW020288,


N25033, AI540458,


AI582932, AI289310,


AI289791, AL118620,


AL036705, AI685080,


AA284380, N81195,


AW161202, AW020381,


AI927233, AW079432,


AI589428, AI697464,


AI625226, AI654258,


AA001397, 841605,


AW020693, AW025279,


AL046466, AI590043,


AI638644, AW 130362,


AI783569, AI628325,


AI440238, AW327693,


AW 193467, AI887430,


AI263584, AA743354,


AI473536, AI344935,


AA761573, AW008737,


262


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI582966, AI866465,


AI613038, AL046454,


AI564259, AL045619,


AI469764, AL046618,


AI432570, AI261589,


AW088628, AW020095,


AI348897, AI345688,


AW 172982, AI699823,


AW089844, AI815239,


AI539847, AI524654,


AL045413, AW268293,


AA975952, AI863241,


AI608936, AI628015,


w AL040241, AW089006,


AI345415, AI798456,


820540, AW020710,


AW075382, N98597,


AI282669, AW021717,


AI582912, AW083572,


AW263709, AI567866,


AL118781, AI584130,


AA678484, AW 117675,


AI919500, AI680467,


AA508692, AW072413,


AL041996, AI866469,


AI799189, AI280607,


AW 194185, AA947065,


AI336565, AI452560,


AI885949, AI049733,


AI334893, AI004911,


AI491710, AI148113,


AW005029, AI114703,


AI633314, AI282319,


AI868204, AA503384,


AI370322, AI612885,


AW006032, AL042382,


AA693347, AI619820,


AI499279, AI610895,


AI521005, AI284509,


AI289542, AI690948,


AI366959, AI309306,


AI623662, AI635813,


AA857847, AI919600,


AI866090, AI523521,


AW167083, AA587590,


AI624548, AW022682,


AI610446, AI610399,


AW079699, AL041150,


AI355779, AI445877,


263


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW079572, AI370623,


AI254226, AI761468,


AF047716, E01314,


AF061943, AL137271,


AL122093, AL117578,


I48978, I89947, AJ012582,


AL049466, AF015958,


X62580, AL133088,


AL137258, AL110224,


AF055917, AL137294,


AL122098, AJ010277,


A93350, AL023657,


M80340, AF169154,


AF120268, S83440, E02221,


AR038854, L13297,


A07647, AF113019,


AR068466, AC004200,


A08907, M19658, U77594,


AL137267, A58524,


AF159148, A58523,


AF028823, AL133558,


M27260, AF177401,


AL137656, I32738,


AL049276, X80340,


A08910, AF030165,


U89295, X72889, E08516,


A18777, A08909;


AF 102166, A08913,


AF150103, AL137658,


A52563, AF137367,


AL137521, AR068753,


A08908, AL049339,


AF094480, A08912,


569510, AF026008,


A08911, L24896, I80062,


AL080124, I89931,


AF067790, AL137557,


X79812, X87582, U92068,


S77771, AL110221,


AF090886, AL133062,


U57352, Y14634,


AL137461, AL137479,


568736, I48979, I49625,


I41145, X66862, E12580,


E12579, AL110225, S76508,


D16301, AL137555,


X15132, X97332,


AL133084, U75370,


AL109672, I33392,


264


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF107847, AL050172,


AF076633, AF132676,


AF017790, AF106657,


AL133606, AF124435,


Y11587, L04859, I89934,


AF061836, AL137463,


X84990, E06788, E06790,


E06789, AR050959,


AF175903, AJ238278,


AF 151109, AF201468,


AL050092, AB030279,


X96540, AL110280,


U49908, AF 115410,
I29004,


X66417, S54890,


AF078844, AL137627,


Y10080, AF065135,


X66975, X82434, I17544,


AL137711, AL133075,


X06146, AF199027,


AL110222, U79523,


AL137550, A23630,


AL122110, S75997,


AR020905, AF207750,


AF017437, AF067728,


A65341, AL122050,


AL117587, AL137459,


AJ006417, AF124728,


AF030513, AL050393,


AF057300, 297214,


AF057299, X83508,


X99226, A70386,


AL133560, E12806,


AF013214, L40363,


AL133067, AR060156,


A07588, AF036941,


A77033, A77035,


AF051325, A76335,


AR029490, AF126488,


AL096720, AF004162,


AL080137, A90832,


Y00093, AL137526,


AL137716, AF026816,


AF 106945, AF 111112,


X98066, AL110196,


AL080159, AL117416,


X52128, AF153205,


U55017, E15324, X67688,


AF106697, AL080139,


AL050149, L31396,


265


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AF158248, AL137254,
AF061981, AF090896,
U78525, AL050155,
AL133619, AL137548,
AL049452, L31397,
AF114168, AL110199,
AL008706, AF113691,
w A15345, X63410,
AF031903, AF 100931,
AF082526, AL133080,
D44497, AF054599,
D89079, A59344, 272491,
AF111851, U53505,
AB025103, AF176651,
and
AF200464.


HE8UT58 177 973153 1 - 1090 15 - 1104AL079991, AW409774,


AA404732, AA782257,


AI808909, AA099036,


AA677654, AA405418,


AW341107, AW409667,


AA659407, AA503263,


AI433410, AI885266,


AI341147, AA905366,


AI692534, AI027623,


AA468344, AA923181,


AA778251, AI681129,


AA533117, AI986154,


AA099037, AI955317,


AI276678, H29006,


AA404602, AI347168,


AA371047, AI420387,


AW 135263, AA868124,


T12623, H09118,


AA430277, AA365338,


T08839, AA813337,


H29109, H 11272,


AW137535, AB414534,


AF116574, AF116573,
and


AC032004.


HIBCN93 178 1178130 1 - 1492 15 - 1506AI056401, AW072652,


AI885072, AW205916,


AI879122, AI885524,


N34233, AI953626,


AI768363, AI500165,


AI887770, AI651798,


AA393235, N35730,


AW297174, AI802927,


AW271854, 856346,


AA249118, AI377463,


266


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AW070857, AI824909,


N53527, AA948310,


AI206861, AA572777,


AA570002, AA814424,


N25635, AA255602,


AI478500, N25539,


AA634056, H07018,


N28490, AA416965,


N34136, N34013,


AA902860, AA255576,


AW029208, AI690664,


AI198003, H99526,
H05467,


N49189, AI190195,


AI864484, N30121,
H99763,


AI024777, AA379362,


AA262183, AI191172,


886778, AA721016,


AA350164, 813979,


AI963889, AA339761,


817378, N49183,


AA262707, T33288,


842616, H99527, T16329,


AI423712, AI952318,


D62700, AI267479,
N92737,


W20356, AA385524,


T28424, AA370138,


AA279758, N24571,


840039, AI351820,


AI674497, AA864521,


T35226, D57142, 856257,


N50244, AI124956,


AF143235, and AW628476.


HDPBI30 179 974711 1 - 2911 15 - 2925 AA714520, N78665,


W15172, AL134531,


AA074818, AI251157,


AI311635, AA079403,


AW 130754, AI935943,


AF083955, AC005015,


AL034423, AP000030,


AC002992, AC004216,


AC003013, U91321,


AC003684, AC002528,


AL117258, AL021155,


AP000045, AF053356,


AL033521, AC004598,


U91326, AL035072,


AD000091, U82668,


AC012384, L44140,


AF006752, AL034350,


267


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AC006039, AC005756,


AC005072, AL034429,


AC002352, AC005682,


AC003663, AC005049,


AC007298, AC005620,


AC004887, AL117694,


AC005911, AC007688,


AC006014, AC004797,


AL031186, AL031283,


AC004963, L47234,


284466, AC004125,


AC005529, AL031293,


AC006276, AL034400,


AC004099, AC005089,


AL049871, AC004893,


AL080243, AC007021,


AL049712, AC007993,


AC006581, AC005837,


AF139813, M13792,


AC005086, AL096791,


AJ251973, AC002301,


AC006139, AC005488,


L78810, AC006115,


AC004966, AC006538,


293244, AC004834,


AL049570, AC004084,


AP000113, AP000251,
and


AC005696.


HLTGA03 180 974851 1 - 890 15 - 904 AI719655, AW082191,


AA761093, AA576454,


AI804392, AA505065,


AA641512, AI220630,
and


AI189378.


HFXJI27 181 971046 1 - 427 15 - 441 AI335925, AI890750,


AA187638, N39062,


AI268071, D79411,
N75622,


N22381, H13284, 843578,


F10497, 826373, AI625385,


AI220697, 821997,


AA932204, AI493179,


AI373111, AA909963,


AI476063, AA909961,


AI888010, AI619918,


AA976881, AW339040,


AI432041, AA015789,


AI049514, AI355877,


AI888471, N59174,


AI679618, AI373318,


AA670204, AI358468,


268


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AI336216, AI266210,


AA947133, 833792,


AW081307, AA610540,


AA928678, AA922947,


AA922946, AI817788,


AI245931, AA948492,


AA044223, T63607,


AI433872, AW269052,


AI587384, AA827770,


AW182646, AI127888,


H89128, AI268557,
D62713,


T63317, 835227,


AA018661, AW023089,


w N67948, T62565, H01498,


821841, AW151373,


D29213, AW022288,


AA678512, and AB023187.


TABLE 4
Code Description Tissue Organ Cell DiseaseVector
Line



AR022a Heart a Heart


AR023a Liver a Liver


AR024a mamma land a mamma land


AR025a Prostate a Prostate


AR026a small intestinea small intestine


AR027a Stomach a Stomach


AR028Blood B cells Blood B cells


AR029Blood B cells Blood B cells
activated activated


AR030Blood B cells Blood B cells
resting restin


AR031Blood T cells Blood T cells
activated activated


AR032Blood T cells Blood T cells
restin restin


AR033brain Grain


AR034breast breast


AR035breast cancer breast cancer


AR036Cell Line CAOV3Cell Line
CAOV3


AR037cell line PA-1 cell line
PA-1


AR038cell line transformedcell line
transformed


AR039colon colon


AR040colon (9808co65R)colon (9808co65R)


AR041colon 9809co15)colon(9809co15)


AR042colon cancer colon cancer


AR043colon cancer colon cancer
(9808co64R) 9808co64R)


AR044colon cancer colon cancer
9809co14 9809co14


AR045corn clone 5 corn clone
5


AR046corn clone 6 corn clone
6


AR047com clone2 com clone2


AR048corn clone3 corn clone3


AR049Corn Clone4 Com Clone4


269


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WO 01/55163 PCT/USO1/01358
AR050Donor 11 B CellsDonor II
24hrs B Cells


24hrs


AR051Donor II B CellsDonor 11
72hrs B Cells


72hrs


AR052Donor II B-CellsDonor 1l
24 hrs. B-Cells
24


hrs.


AR053Donor 11 B-CellsDonor II
72hrs B-Cells


72hrs


AR054Donor 11 RestingDonor II
B Cells Resting
B


Cells


AR055Heart Heart


AR056Human Lung (clonetech)Human Lung


(clonetech)


AR057Human Mammary Human Mammary


(clontech) (clontech)


AR058Human Thymus Human Thymus


clonetech) (clonetech)


AR059Jurkat (unstimulated)Jurkat


(unstimulated)


AR060Kidne Kidne


AR061Liver Liver


AR062Liver (Clontech)Liver (Clontech)


AR063Lymphocytes Lymphocytes
chronic


lymphocytic chronic lymphocytic
leukaemia


leukaemia


AR064Lymphocytes Lymphocytes
diffuse large


B cell lymphomadiffuse large
B cell


lym homa


AR065Lymphocytes Lymphocytes
follicular


I homa follicular
1 homa


AR066normal breast normal breast


AR067Normal Ovarian Normal Ovarian


4004901 (4004901)


AR068Normal Ovary NotTrtal
95086045 Ovary


95086045


AR069Normal Ovary Normal Ovary
97016208


97016208


AR070Normal Ovary Normal Ovary
98066005


98066005


AR071Ovarian Cancer Ovarian Cancer


AR072Ovarian Cancer Ovarian Cancer


(97026001 (97026001)


AR073Ovarian Cancer Ovarian Cancer


97076029) (97076029)


AR074Ovarian Cancer Ovarian Cancer


9804601 I 98046011
)


AR075Ovarian Cancer Ovarian Cancer


98066019 98066019)


AR076Ovarian Cancer Ovarian Cancer


(98076017) (98076017)


AR077Ovarian Cancer Ovarian Cancer


98096001) 98096001)


AR078ovarian cancer ovarian cancer
15799


15799


AR079Ovarian Cancer Ovarian Cancer


17717AID 17717AID


AR080Ovarian Cancer Ovarian Cancer


4004664B1 400466481


AR081Ovarian Cancer Ovarian Cancer


4005315A1 4005315A1


AR082ovarian cancer ovarian cancer
94127303


94127303


AR083Ovarian Cancer Ovarian Cancer
96069304


270


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WO 01/55163 PCT/USO1/01358
96069304


AR084Ovarian Cancer Ovarian Cancer
97076029


97076029


ARO85Ovarian Cancer Ovarian Cancer
98076045


98076045


AR086ovarian cancer ovarian cancer
98096001


98096001


AR087Ovarian Cancer Ovarian Cancer


9905C032RC 9905C032RC


AR088Ovarian cancer Ovarian cancer
9907 C00 9907


3rd C00 3rd


AR089Prostate Prostate


AR090Prostate (clonetech)Prostate
(clonetech)


AR091rostate cancer rostate cancer


AR092prostate cancerprostate
# 15176 cancer


#15176


AR093prostate cancerprostate
#15509 cancer


#15509


AR094prostate cancerprostate
# 15673 cancer


#15673


AR095Small IntestineSmall Intestine
(Clontech)


Clontech)


AR096S teen Spleen


AR097Thymus T cells Thymus T
activated cells


activated


AR098Thymus T cells Thymus T
resting cells


resting


AR099Tonsil Tonsil


AR100Tonsil geminal Tonsil geminal
center


centroblast center centroblast


AR101Tonsil germinalTonsil germinal
center B


cell center B
cell


AR102Tonsil lym h Tonsil lym
node h node


AR103Tonsil memory Tonsil memory
B cell B


cell


AR104Whole Brain Whole Brain


AR105Xeno aft ES-2 Xeno aft
ES-2


AR106Xeno aft SW626 Xeno aft
SW626


H0002Human Adult Human Adutt Heart Uni-ZAP
Heart Heart


XR


H0004Human Adult Human Adult Spleen Uni-ZAP
Spleen


S teen XR


H0008Whole 6 Week Uni-ZAP
Old


Emb o XR


H0009Human Fetal Uni-ZAP
Brain


XR


H0011Human Fetal Human Fetal Kidney Uni-ZAP
Kidney Kidney


XR


H0012Human Fetal Human Fetal Kidney Uni-ZAP
Kidney Kidney


XR


H0013Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old


Emb o Emb o XR


H0014Human Gall BladderHuman Gall Gall Bladder Uni-ZAP
Bladder


XR


HOOlSHuman Gall Bladder,Human Gall Gall Bladder Uni-ZAP
Bladder


fraction II XR


H0017Human Greater Human Greaterperitoneum Uni-ZAP
Omentum


Omentum XR


H0018Human Greater Human Greaterperitoneum Uni-ZAP
Omentum,


fII remake Omentum XR


H0020Human HippocampusHuman Brain Uni-ZAP


Hi ocam us XR


H0024Human Fetal Human Fetal Lung ~ ~ Uni-ZAP
Lung III Lung


271


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WO 01/55163 PCT/USO1/01358
XR


H0030Human Placenta Uni-ZAP


XR


H0031Human Placenta Human PlacentaPlacenta Uni-ZAP


XR


H0032Human Prostate Human ProstateProstate Uni-ZAP


XR


H0033Human PituitaryHuman Pituitary Uni-ZAP


XR


H0036Human Adult Human Adult Small Uni-ZAP
Small Small Int.


Intestine Intestine XR


H0037Human Adult Human Adult Small pBluescript
Small Small Int.


Intestine Intestine


H0038Human Testes Human TestesTestis Uni-ZAP


XR


H0039Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor


Tumor XR


H0040~ Human Testes Human TestesTestis diseaseUni-ZAP
Tumor


Tumor XR


H0041Human Fetal Human Fetal Bone Uni-ZAP
Bone Bone


XR


H0042Human Adult Human Adult Lung Uni-ZAP
Pulmonary


Pulmona XR


H0046Human EndometrialHuman EndometrialUterus diseaseUni-ZAP


Tumor Tumor XR


H0050Human Fetal Human Fetal Heart Uni-ZAP
Heart Heart


XR


H0051Human HippocampusHuman Brain Uni-ZAP


Hi pocam XR
us


H0052Human CerebellumHuman CerebellumBrain Uni-ZAP


XR


H0056Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein,


Endo. remake Vein Endothelialvein XR


Cells


H0057Human Fetal Uni-ZAP
Spleen


XR


H0059Human Uterine Human UterineUterus diseaseLambda
Cancer


Cancer ZAP II


H0060Human Macro Human Macro Blood Cell Bluescri
ha a ha a Line t


H0063Human Thymus Human ThymusThymus Uni-ZAP


XR


H0068Human Skin TumorHuman Skin Skin diseaseUni-ZAP
Tumor


XR


H0069Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells T-Cells Line


XR


H0081Human Fetal Human Fetal Skin Uni-ZAP
Epithelium Skin


Skin XR


H0083HUMAN JURKAT Jurkat Cells Uni-ZAP
'


MEMBRANE BOUND XR


POLYSOMES


H0085Human Colon Human Colon Lambda


ZAP II


H0087Human Th us Human Th Bluescri
us t


H0090Human T-Cell T-Cell LymphomaT-Cell diseaseUni-ZAP
Lymphoma


XR


H0098Human Adult Human Adult Liver Uni-ZAP
Liver, Liver


subtracted XR


H0099Human Lung Cancer,Human Lung Lung pBluescript
Cancer


subtracted


HO100Human Whole Human Whole Embryo Uni-ZAP
Six Week Six


Old Emb o Week Old XR
Emb o


H0103Human Fetal Human Fetal Brain Uni-ZAP
Brain, Brain


subtracted XR


272


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H0123 Human Fetal Human FetalBrain Uni-ZAP
Dura Mater Dura


Mater XR


'H0124Human Human Sk Muscle diseaseUni-ZAP


Rhabdom osarcomaRhabdom XR
osarcoma


H0125 Cem cells cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line


treated Treated XR
Cem, Jurkat,


Ra~i, and
Su t


H0131 LNCAP + o.3nM LNCAP Cell Prostate Cell Uni-ZAP
81881 Line Line


XR


H0132 LNCAP + 30nM LNCAP Cell Prostate Cell Uni-ZAP
81881 Line Line


XR


H0134 Raji Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line


treated Treated XR
Cem, Jurkat,


Ra'i, and
Su t


H0135 Human Synovial Human SynovialSynovium Uni-ZAP
Sarcoma


Sarcoma XR


H0136 Supt Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line


'treated Treated XR
Cem, Jurkat,


Ra~i, and
Su t


H0141 Activated T-Cells,Activated Blood Cell Uni-ZAP
12 hrs. T-Cells Line


XR


H0144 Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early


Stage Human Stage Human XR


HO150 Humari EpididymusEpididymis Testis Uni-ZAP


XR


H0156 Human Adrenal Human AdrenalAdrenal diseaseUni-ZAP
Gland


Tumor Gland TumorGland XR


H0163 Human Synovium Human SynoviumSynovium Uni-ZAP


XR


H0164 Human Trachea Human TracheaTrachea diseaseUni-ZAP
Tumor


Tumor XR


H0166 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer,


Sta a B2 fractionCancer, XR
stage B2


H0167 Activated T-Cells,Activated Blood Cell Uni-ZAP
24 hrs. T-Cells Line


XR


H0169 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer,


Stage C fractionCancer, XR
stage C


H0170 12 Week Old Twelve WeekEmbryo Uni-ZAP
Early Stage Old


Human Earl Sta XR
a Human


H0171 12 Week Old Twelve WeekEmbryo Uni-ZAP
Early Stage Old


Human, II Earl Sta XR
a Human


H0172 Human Fetal Human FetalBrain Lambda
Brain, Brain


random rimed ZAP
II


H0176 CAMA1 Ee Cell CAMA1 Ee Breast Cell Uni-ZAP
Line Cell Line


Line XR


H0178 Human Fetal Human FetalBrain Uni-ZAP
Brain Brain


XR


H0179 Human NeutrophilHuman NeutrophilBlood Cell Uni-ZAP
Line


XR


H0181 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast


Cancer Breast Cancer XR


H0187 Resting T-Cell T-Cells Blood Cell Lambda
Line


ZAP
II


H0188 Human Normal Human NormalBreast Uni-ZAP
Breast


Breast XR


H0191 Human ActivatedHuman Blood Cell Uni-ZAP
Line


Macrophage (LPS),Macrophage/Monoc XR
thiour


es


H0194 Human Cerebellum,Human CerebellumBrain pBluescript


subtracted


H0196 Human Cardiomyopathy,Human Heart Uni-ZAP


subtracted Cardiom XR
o ath


H0200 Human Greater Human Greatereritoneum Uni-ZAP
Omentum,


273


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
fract II remake,Omentum XR


H0201Human Hippocampus,Human Brain pBluescript


' subtracted Hi ocam us


H0207LNCAP, differentialLNCAP Cell Prostate Cell pBluescript
Line Line


ex ression


H0208Early Stage Human Fetal Lung pBluescript
Human Lung, Lung


subtracted


H0213Human Pituitary,Human Pituitary Uni-ZAP


subtracted ' XR


H0214Raji cells, CyclohexamideBlood Cell pBluescript
cyclohexamide Line


treated, subtractedTreated Cem,
Jurkat,


Raji, and
Su t


H0222Activated T-Cells,Activated Blood Cell Uni-ZAP
8 hrs, T-Cells Line


subtracted XR


H0233Human Fetal Human Fetal Heart pBluescript
Heart, Heart


Differential
(Adult-


S ecific)


H0244'Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old


Embryo, subtractedEmbryo XR


H0250Human ActivatedHuman Monocytes Uni-ZAP


Monoc es XR


H0251Human ChondrosarcomaHuman Cartilage diseaseUni-ZAP


Chondrosarcoma XR


H0252Human OsteosarcomaHuman Bone diseaseUni-ZAP


Osteosarcoma XR


H0253Human adult Human Adult Testis Uni-ZAP
testis, large Testis


inserts XR


H0254Breast Lymph Breast LymphLymph Uni-ZAP
node cDNA Node Node


library XR


H0255breast lymph Breast LymphLymph Lambda
node CDNA Node Node


librar ZAP II


H0257HL-60, PMA 4H HL-60 Cells,Blood Cell Uni-ZAP
PMA Line


stimulated XR
4H


H0261H. cerebellum, Human CerebellumBrain Uni-ZAP
Enzyme


subtracted XR


H0263human colon Human Colon Colon diseaseLambda
cancer


Cancer ZAP II


H0264human tonsils Human TonsilTonsil Uni-ZAP


XR


H0265Activated T-CellT-Cells Blood Cell Uni-ZAP
Line


(l2hs)/Thiouridine XR


labelledEco


H0266Human MicrovascularHMEC Vein Cell Lambda
Line


Endothelial ZAP II
Cells, fract.
A


H0268Human UmbilicalHUVE Cells UmbilicalCell Lambda
Vein Line


Endothelial vein ZAP II
Cells, fract.
A


H0269Human UmbilicalHUVE Cells UmbilicalCell Lambda
Vein Line


Endothelial vein ZAP II
Cells, fract.
B


H0271Human Neutrophil,Human NeutrophilBlood Cell Uni-ZAP
- Line


Activated Activated XR


H0272HUMAN TONSILS, Human TonsilTonsil Uni-ZAP


FRACTION 2 XR


H0282HBGB"s differentialHuman PrimaryBreast Uni-ZAP


consolidation Breast Cancer XR


H0284Human OB MG63 Human Bone Cell Uni-ZAP
control Line


fraction I Osteoblastoma XR


MG63 cell
line


H0286Human OB MG63 Human Bone Cell Uni-ZAP
treated Line


(10 nM E2) fractionOsteoblastoma XR
I


MG63 cell
line


H0292Human OB HOS Human Bone Cell Uni-ZAP
treated Line


(10 nM E2) fractionOsteoblastoma XR
I HOS


cell line


274


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
H0294 Amniotic Cells Amniotic PlacentaCell Uni-ZAP
- TNF Cells - Line


induced TNF induced XR


' H0295Amniotic Cells Amniotic PlacentaCell Uni-ZAP
- Primary Cells - Line


Culture Prima Culture XR


H0305 CD34 positive CD34 PositiveCord ZAP
cells (Cord Cells Blood


Blood) Express


H0306 CD34 depleted CD34 DepletedCord ZAP
Buffy Coat Blood


(Cord Blood) Buffy Coat Express
(Cord


Blood)


H0309 Human Chronic Synovium, Synovium diseaseUni-ZAP
Synovitis Chronic


Synovitis/ XR


Osteoarthritis


H0316 HUMAN STOMACH Human StomachStomach Uni-ZAP


XR


H0318 HUMAN B CELL Human B CellLymph diseaseUni-ZAP
Node


LYMPHOMA L homa XR


H0327 human corpus Human CorpusBrain Uni-ZAP
colosum


Callosum XR


H0328 human ovarian Ovarian CancerOvary diseaseUni-ZAP
cancer


XR


H0329 DermatofibrosarcomaDermatofibrosarcomSkin diseaseUni-ZAP


Protuberance a Protuberans XR


H0331 Hepatocellular HepatocellularLiver diseaseLambda
Tumor


Tumor ZAP II


H0333 HemangiopericytomaHemangiopericytomBlood diseaseLambda
vessel


a ZAP II


H0334 Kidney cancer Kidney CancerKidney diseaseUni-ZAP


XR


H0341 Bone Marrow Bone Marrow Bone Cell Uni-ZAP
Cell Line Cell Marrow Line


RS4;1 1 ) Line RS4;11 XR


H0342 Lingual Gyrus Lingual GyrusBrain Uni-Zap


XR


H0344 Adipose tissue Adipose - Uni-ZAP
(human) 6825A


human XR


H0345 SKIN Skin - 4000868HSkin Uni-ZAP


XR


H0346 Brain-medulloblastomaBrain Brain diseaseUni-ZAP


(Medul loblastoma)- XR


9405C006R


H0351 Glioblastoma GlioblastomaBrain diseaseUni-ZAP


XR


H0352 wilm"s tumor Wilrri's diseaseUni-ZAP
Tumor


XR


H0354 Human LeukocytesHuman LeukocytesBlood Cell pCMVSport
Line 1


H0355 Human Liver Human Liver, pCMVSport


normal Adult 1


H0356 Human Kidney Human KidneyKidney pCMVSport
1


H0357 H. Normalized Human Fetal Liver Uni-ZAP
Fetal Liver


Liver, 11 XR


H0361 Human rejected Human Rejected diseasepBluescript
kidney


Kidney


H0369 H. Atrophic Atrophic Uni-ZAP
Endometrium


Endometrium XR
and


m ometrium


H0370 H. Lymph node Lymph node diseaseUni-ZAP
breast with


Cancer Met. Breast XR
Cancer


H0372 Human Testes Human TestesTestis pCMVSport
1


H0373 Human Heart Human Adult Heart pCMVSport
Heart 1


H0375 Human Lun Human Lun pCMVSport


275


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
I


H0379 Human Tongue, Human Tongue S ortl
frac 1


' H0383Human Prostate Human Prostate Uni-ZAP
BPH, re-


excision BPH XR


H0390 Human Amygdala Human Amygdala diseasepBluescript


De ression, De ression
re-excision


H0391 H. Meniin ima, Human Menin brain S ortl
M6 ima


H0392 H. Meningima, Human Meningimabrain S ortl
M1


H0393 Fetal Liver, Human Fetal Liver Bluescri
subtraction Liver t
l1


H0394 A-14 cell line Redd-Stemberg ZAP
cell


Ex ress


H0395 A1-CELL LINE Redd-Stemberg ZAP
cell


Ex ress


H0398 Human Newborn Human Newborn pBluescript
Bladder


Bladder


H0399 Human Kidney Human Kidney Lambda
Cortex, re-


rescue Cortex ZAP II


H0400 Human Striatum Human Brain,Brain Lambda


De ression, Striatum ZAP II
re-rescue De ression


H0402 CD34 depleted CD34 DepletedCord ZAP
Buffy Coat Blood


(Cord Blood), Buffy Coat Express
re-excision (Cord


Blood)


H0404 H. Umbilical HUVE Cells UmbilicalCell Uni-ZAP
Vein Line


endothelial vein XR
cells,


uninduced


H0408 Human kidney Human Kidney pBluescript
Cortex,


subtracted Cortex


H0409 H. Striatum Human Brain,Brain pBluescript
Depression, ,


subtracted Striatum
De ression


H0411 H Female Bladder,Human FemaleBladder pSportl
Adult


Adult Bladder


H0412 Human umbilicalHUVE Cells UmbilicalCell pSportl
vein Line


endothelial vein
cells, IL-4


induced


H0413 Human UmbilicalHUVE Cells UmbilicalCell pSportl
Vein Line


Endothelial vein
Cells,


uninduced


H0414 Ovarian Tumor Ovarian Tumor,Ovary diseasepSportl
I, OV5232


OV5232


H0415 H. Ovarian Tumor,Ovarian Tumor,Ovary diseasepCMVSport
II,


OV5232 OV5232 2.0


H0416 Human Neutrophils,Human NeutrophilBlood, Cell pBluescript
- Line


Activated, re-excisionActivated


H0421 Human Bone Marrow,Bone Marrow pBluescript
re-


excision


H0422 T-Cell PHA 16 T-Cells Blood Cell S ortl
hrs Line


H0423 T-Cell PHA 24 T-Cells Blood Cell S ortl
hrs Line


H0427 Human Adipose Human Adipose, pSportl
left


hi 1i oma


H0428 Human Ovary Human Ovary Ovary pSportl


Tumor


H0429 K562 + PMA (36 K562 Cell cell Cell ZAP
hrs),re- line line Line


excision Ex ress


H0431 H. Kidney Medulla,Kidney medullaKidney pBluescript
re-


excision


H0432 H. Kidne P amidKidne amids Kidne Bluescri
t


H0435 Ovarian Tumor Ovarian Tumor,Ovary pCMVSport
10-3-95


OV350721 2.0


H0436 Restin T-Cell T-Cells Blood Cell S ortl
Libra ,II Line


H0437 H Umbilical HUVE Cells UmbilicalCell Lambda
Vein Line


Endothelial vein ZAP II
Cells, frac
A,


re-excision


H0438 H. Whole Brain Human Whole ZAP
#2, re- Brain


276


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
excision #2 Ex ress


H0441H. Kidney Cortex,Kidney cortexKidney pBluescript


' subtracted


H0442H. Striatum Depression,Human Brain,Brain pBluescript


subt 11 Striatum
Depression


H0443H. Adipose, subtractedHuman Adipose, pSportl
left


hi Ii oma


H0444Spleen metastic Spleen, Spleen diseasepSportl
melanoma Metastic


malignant


melanoma


H0445Spleen, Chronic Human Spleen,Spleen diseasepSportl
CLL


lym hocytic leukemia


H0449CD34+ cell, I CD34 ositive S ortl
cells


H0453H. Kidney Pyramid,Kidney pyramidsKidney pBluescript
,


subtracted


H0457Human Eosino Human Eosino S ortl
hils hils


H0459CD34+cells, l1, CD34 positive pCMVSport
cells


FRACTION 2 2.0


H0478Salivary Gland, Human SalivarySalivary pSportl
Lib 2


Gland land


H0483Breast Cancer Breast Cancer pSportl
cell line, Cell


MDA 36 line, MDA
36


H0484Breast Cancer Breast Cancer pSportl
Cell line, Cell


angiogenic line, Angiogenic,


36T3


H0485Hodgkin"s LymphomaHodgkin"s diseasepCMVSport
I


L homa I 2.0


H0486Hodgkin"s LymphomaHodgkin"s diseasepCMVSport,
II


Lymphoma 2.0
II


H0487Human Tonsils, Human Tonsils pCMVSport
lib 1


2.0


H0488Human Tonsils, Human Tonsils pCMVSport
Lib 2


2.0


H0494Keratinocyte Keratinocyte pCMVSport


2.0


H0497HEL cell line HEL cell HEL pSportl
line


92.1.7


H0506Ulcerative ColitisColon Colon S ortl


H0509Liver, Hepatoma Human Liver,Liver diseasepCMVSport


He atoma,atient 3.0
8


HO510Human Liver, Human Liver,Liver pCMVSport
normal


normal, 3.0
Patient
# 8


H0517Nasal polyps Nasal polyps pCMVSport


2.0


HOS pBMC stimulated pBMC stimulated pCMVSport
18 w/ poly


I/C with of 3.0
I/C


H0519NTERA2, control NTERA2, pCMVSport


Teratocarcinoma 3.0


cell line


H0520NTERA2 + retinoicNTERA2, pSportl
acid,


14 days Teratocarcinoma


cell line


H0521Primary DendriticPrimary pCMVSport
Cells, Dendritic


lib 1 cells 3.0


H0522Primary DendriticPrimary pCMVSport
Dendritic


cells,frac 2 cells 3.0


H0529Myoloid ProgenitorTF-1 Cell pCMVSport
Cell Line;


Line Myoloid 3.0
progenitor


cell line


H0538Merkel Cells Merkel cellsL h node S ortl


H0539Pancreas Islet Pancreas Pancreas diseasepSportl
Cell Tumor Islet Cell


Tumour


H0540Skin burned Skin leg Skin ~ ~ pSportl
burned


277


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
H0542 T Cell helper Helper T pCMVSport
I cell


3.0


' H0543T cell helper Helper T pCMVSport
II cell


3.0


H0544 Human endometrialHuman endometrial pCMVSport


stromal cells stromal cells 3.0


H0545 Human endometrialHuman endometrial pCMVSport


'stromal cells-treatedstromal cells-treated 3.0
with


rogesterone with rope


H0546 Human endometrialHuman endometrial pCMVSport


stromal cells-treatedstromal cells-treated 3.0
with


estradiol with estra


H0547 NTERA2 teratocarcinomaNTERA2, pSportl


cell line+retinoicTeratocarcinoma
acid (14


da s) cell line


H0549 H. Epididiymus,Human Uni-ZAP
caput &


corpus Epididiymus, XR
caput


and co us


HO550 H. Epididiymus,Human Uni-ZAP
cauda


E ididiymus, XR
cauda


HO551 Human Thymus Human Thymus pCMVSport
Stromal


Cells Stromal Cells 3.0


H0553 Human Placenta Human Placenta pCMVSport


3.0


HOS55 Rejected Kidney,Human RejectedKidney diseasepCMVSport
lib 4


Kidne 3.0


H0556 Activated T- T-Cells Blood Cell Uni-ZAP
Line


cell( 12h)/Thiouridine-re- XR


excision


H0559 HL-60, PMA 4H, HL-60 Cells,Blood Cell Uni-ZAP
re- PMA Line


excision stimulated XR
4H


H0560 KMH2 KMH2 pCMVSport


3.0


H0561 L428 L428 pCMVSport


3.0


H0570 Human Fetal Human Fetal pCMVSport
Brain, Brain


normalized CSOOH 2.0


H0571 Human Fetal Human Fetal pCMVSport
Brain, Brain


normalized CSOOHE 2.0


H0572 Human Fetal Human Fetal pCMVSport
Brain, Brain


normalized AC5002 2.0


H0574 Hepatocellular HepatocellularLiver diseaseLambda
Tumor; re-


excision Tumor ZAP II


H0575 Human Adult Human Adult Lung Uni-ZAP


Pulmona ;re-excisionPulmonary XR


H0576 Resting T-Cell;T-Cells Blood Cell Lambda
re- Line


excision ZAP II


H0580 Dendritic cells,Pooled dendritic pCMVSport
pooled


cells 3.0


H0581 Human Bone Marrow,Human Bone Bone pCMVSport
Marrow


treated Marrow 3.0


H0583 B Cell lymphomaB Cell LymphomaB Cell diseasepCMVSport


3.0


H0584 Activated T-cells,Activated Blood Cell Uni-ZAP
24 T-Cells Line


hrs,re-excision XR


H0585 Activated T-Cells,l2Activated Blood Cell Uni-ZAP
T-Cells Line


hrs,re-excision XR


H0586 Healing groin healing groingroin diseasepCMVSport
wound, 6.5


hours post incisionwound, 6.5 3.0
hours


ost incision
- 2/


H0587 Healing groin Groin-2/19/97groin diseasepCMVSport
wound; 7.5


hours ost incision 3.0


H0589 CD34 positive CD34 PositiveCord ZAP
cells (cord Cells Blood


278


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
blood),re-ex Ex ress


H0590 Human adult Human Adult Small Uni-ZAP
small Small Int.


' intestine,re-excisionIntestine XR


H0591 Human T-cell T-Cell LymphomaT-Cell diseaseUni-ZAP


I homa;re-excision XR


H0592 Healing groin HGS wound diseasepCMVSport
wound - healing


zero hr post-incisionproject; 3.0
abdomen


control)


H0593 Olfactory Olfactory pCMVSport
epithelium


epithelium;nasalcavityfrom roof 3.0
of left


nasal tacit


H0594 Huinan Lung Human Lung Lung diseaseLambda
Cancer;re- Cancer


excision ZAP
11


H0595 Stomach cancer Stomach Cancer diseaseUni-ZAP
-


human);re-excision5383A (human) XR


H0596 Human Colon Human Colon Colon Lambda
Cancer;re-


excision Cancer ZAP
II


H0597 'Human Colon; Human Colon Lambda
re-excision


ZAP
II


H0598 Human Stomach;re-Human StomachStomach Uni-ZAP


excision XR


H0599 Human Adult Human Adult Heart Uni-ZAP
Heartre- Heart


excision XR


H0600 Healing AbdomenAbdomen diseasepCMVSport


wound;70&90 3.0
min post


incision


H0604 Human Pituitary,Human Pituitary pBluescript
re-


excision


H0606 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast


Cancer;re-excisionBreast Cancer XR


H0608 H. Leukocytes, H.Leukocytes pCMVSport
control 1


H0613 H.Leukocytes, H.Leukocytes pCMVSport
normalized


cot 5 B 1


H0615 Human Ovarian Ovarian CancerOvary diseaseUni-ZAP
Cancer


Reexcision XR


H0616 Human Testes, Human TestesTestis Uni-ZAP
Reexcision


XR


H0617 Human Primary Human PrimaryBreast diseaseUni-ZA1'
Breast


Cancer ReexcisionBreast Cancer XR


H0618 Human Adult Human Adult Testis Uni-ZAP
Testes, Testis


Lar a Inserts, XR
Reexcision


H0619 Fetal Heart Human Fetal Heart Uni-ZAP
Heart


XR


H0620 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney; Kidney


Reexcision XR


H0622 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor;


Reexcision Tumor XR


H0623 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein;


Reexcision Vein Endothelialvein XR


Cells


H0624 12 Week Early Twelve Week Embryo Uni-ZAP
Stage Old


Human II; ReexcisionEarly Sta XR
a Human


H0625 Ku 812F Baso Ku 812F Baso S ortl
hils Line hils


H0626 Saos2 Cells; Saos2 Cell pSportl
Untreated Line;


Untreated


H0627 Saos2 Cells; Saos2 Cell pSportl
Vitamin D3 Line;


Treated Vitamin D3
Treated


H0628 Human Pre-DifferentiatedHuman Pre- Uni-ZAP


Adipocytes Differentiated XR


Adi oc es


H0630 Human Human Normalized pCMVSport


Leukocytes,normalizedleukocyte 1


279


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
control #4


H0631 Saos2, DexamethosomeSaos2 Cell pSportl
Line;


' Treated Dexamethosome


Treated


H0632 Hepatocellular HepatocellularLiver Lambda
Tumor;re-


excision Tumor ZAP
II


H0633 Lung Carcinoma TNFalpha diseasepSportl
A549 activated


TNFalpha activatedA549--Lung


Carcinoma


H0634 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor, re-


excision Tumor XR


H0635 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells, T-Cells Line


re-excision XR


H0637 Dendritic CellsDentritic pSportl
From cells from


CD34 Cells CD34 cells


H0638 CD40 activated CD40 activated pSportl
monocyte


dendridic cellsmonocyte
dendridic


' cells


H0641 LPS activated . LPS activated pSportl
derived


dendritic cellsmonocyte
derived


dendritic
cells


H0642 Hep G2 Cells, Hep G2 Cells Other
lambda


libra


H0643 He G2 Cells, He G2 Cells Other
PCR libra


H0644 Human Placenta Human PlacentaPlacenta Uni-ZAP
(re-


excision) XR


H0645 Fetal Heart, Human Fetal Heart Uni-ZAP
re-excision Heart


XR


H0646 Lung, Cancer Metastatic pSportl
(4005313


A3): Invasive squamous
Poorly cell lung


Differentiated carcinoma,
Lung poorly di


Adenocarcinoma,


H0647 Lung, Cancer Invasive diseasepSport
(4005163 poorly 1


B7): Invasive, differentiated
Poorly Diff. lung


Adenocarcinoma,adenocarcinoma


Metastatic


H0648 Ovary, Cancer: Papillary diseasepSportl
(4004562 Cstic


B6) Papillary neoplasm
Serous of low


Cystic Neoplasm,malignant
Low potentia


Mali ant Pot


H0649 Lung, Normal: Normal Lung pSportl
(4005313 ,


B1)


H0650 B-Cells B-Cells pCMVSport


3.0


H0651 Ovary, Normal: Normal Ovary pSport
l


(9805C040R)


H0654 Lung, Cancer: Metastatic Other
(4005313


A3) Invasive Squamous
Poorly- cell lung


differentiated Carcinoma
Metastatic poorly


lung adenoc dif


H0656 B-cells (unstimulated)B-cells pSportl


unstimulated)


H0657 B-cells (stimulated)B-cells (stimulated) S ortl


H0658 Ovary, Cancer 9809C332- Ovary diseasepSportl
Poorly &


(9809C332): differentiateFallopian
Poorly


differentiated Tubes


adenocarcinoma


H0659 Ovary, Cancer Grade II Ovary diseasepSportl
Papillary


(15395A1F): Carcinoma,
Grade II Ovary


Pa illary Carcinoma


H0660 Ovary, Cancer: Poorly differentiated diseasepSportl


(15799A1F) Poorlycarcinoma, ,
ovary


differentiated
carcinoma


H0661 Breast, Cancer:Breast cancer diseasepSportl
(4004943


280


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
AS)


H0662 Breast, Normal:Normal BreastBreast pSportl
-


(400552282) #4005522(B2)


H0663 Breast, Cancer:Breast CancerBreast ' diseasepSportl
(4005522 -


A2) #4005522(A2)


H0664 Breast, Cancer:Breast CancerBreast diseasepSportl


(9806C012R)


H0665 Stromal cells Stromal cells S ortl
3.88 3.88


H0666 Ovary, Cancer: Ovarian Cancer, diseasepSportl
(4004332


A2) Sampte


#4004332A2


H0667 Stromal cells(HBM3.18)Stromal ccll(HBM pSportl


3.18


H0668 stromal cell stromal cell pSportl
clone 2.5 clone


2.5


H0670 Ovary, Cancer(4004650Ovarian Cancer pSportl
-


A3): Well-Differentiated4004650A3


Micropapillary
Serous


Carcinoma


H0671 Breast, Cancer:Breast Cancer- pSportl


(9802C020E) Sample #


9802C020E


H0672 Ovary, Cancer: Ovarian Ovary pSportl
(4004576


A8) Cancer(4004576A8)


H0673 Human Prostate Human ProstateProstate Uni-ZAP
Cancer,


Sta a B2; re-excisionCancer, stage XR
B2


H0674 Human Prostate Human ProstateProstate Uni-ZAP
Cancer,


Sta a C; re-excissionCancer, sta XR
a C


H0675 Colon, Cancer: Colon Cancer pCMVSport


(9808C064R) 9808C064R 3.0


H0677 TNFR de enerateB-Cells PCRII
oli o


H0682 Serous Papillaryserous papillary pCMVSport


Adenocarcinoma adenocarcinoma 3.0


(9606G304SPA3B)


H0683 Ovarian Serous Serous papillary pCMVSport
Papillary


Adenocarcinoma adenocarcinoma, 3.0


stage 3C
(9804601


H0684 Serous PapillaryOvarian Cancer-Ovaries pCMVSport


Adenocarcinoma 98106606 3.0


H0685 Adenocarcinoma Adenocarcinoma pCMVSport
of of


Ovary, Human Ovary, Human 3.0
Cell Line, Cell


# OVCAR-3 Line, # OVCAR-


H0686 Adenocarcinoma Adenocarcinoma pCMVSport
of of


Ovary, Human Ovary, Human 3.0
Cell Line Cell


Line, # SW-626


H0687 Human normal Human normalOvary pCMVSport


ov (#96106215) ova (#96106215) 3.0


H0689 Ovarian Cancer Ovarian Cancer, pCMVSport


#98066019 3.0


H0690 Ovarian Cancer,Ovarian Cancer, pCMVSport
#


97026001 #97026001 3.0


H0691 Normal Ovary, normal ovary, pCMVSport


#971 OG208 #971 OG208 3.0


H0693 Normal ProstateNormal Prostate pCMVSport


#ODQ3958EN Tissue # 3.0


OD 3958EN


H0694 Prostate gland Prostate prostate pCMVSport
gland,


adenocarcinoma adenocarcinoma,gland 3.0


mod/diff,
leason


N0006 Human Fetal Human Fetal
Brain Brain


S0001 Brain frontal Brain frontalBrain Lambda
cortex cortex


ZAP !I


50002 Monocyte activatedMonocyte-activatedblood Cell Uni-ZAP
Line


281


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
XR


50003 Human OsteoclastomaOsteoclastomabone diseaseUni-ZAP


XR


S0004 Prostate Prostate Prostate Lambda
BPH


ZAP II


S0007 Early Stage Human Fetal Uni-ZAP
Human Brain Brain


XR


50010 Human Amygdala Amygdala Uni-ZAP


XR


5001 STROMAL - Osteoclastomabone diseaseUni-ZAP
1


OSTEOCLASTOMA XR


50013 Prostate Prostate prostate Uni-ZAP


XR


50016 Kidney PyramidsKidney pyramidsKidney Uni-ZAP


XR


50021 Whole brain Whole brain Brain ZAP


Ex ress


S0022 Human OsteoclastomaOsteoclastoma Uni-ZAP


Stromal Cells Stromal Cells XR
-


unam lifted


S0026 Stromal cell stromal cellBone Cell Uni-ZAP
TF274 marrow Line


XR


50027 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
serum Line


treated ante XR


S0028 Smooth muscle,controlSmooth musclePulmanaryCell Uni-ZAP
Line


artery XR


S0030 Brain pons Brain Pons Brain Uni-ZAP


XR


S0031 Spinal cord Spinal cord spinal Uni-ZAP
cord


XR


50032 Smooth muscle-ILbSmooth musclePulmanaryCell Uni-ZAP
Line


induced ante XR


50036 Human SubstantiaHuman Substantia Uni-ZAP
Nigra


Ni a XR


S0037 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
ILIb Line


induced artery XR


50038 Human Whole Human Whole ZAP
Brain #2 - Brain


Oli o dT > I.SKb#2 Ex ress


50040 Adipocytes Human Adipocytes Uni-ZAP


from Osteoclastoma XR


50044 Prostate BPH prostate Prostate diseaseUni-ZAP
BPH


XR


50045 Endothelial Endothelial endothelialCell Uni-ZAP
cells-control cell Line


cell-lun XR


S0046 Endothelial-inducedEndothelial endothelialCell Uni-ZAP
cell Line


cell-lun XR


S0049 Human Brain, Human Brain, Uni-ZAP
Striatum


Striatum XR


50050 Human Frontal Human Frontal diseaseUni-ZAP
Cortex,


Schizophrenia Cortex, XR


Schizo hrenia


S0051 Human Human diseaseUni-ZAP


Hypothalmus,SchizophrenHypothalamus, XR


is Schizo hrenia


S0052 neutrophils human neutrophilsblood Cell Uni-ZAP
control Line


XR


S0053 Neutrophils human neutrophilblood Cell Uni-ZAP
tL-1 and LPS Line


induced induced XR


S0106 STRIATUM BRAIN diseaseUni-ZAP


DEPRESSION XR


SO110 Brain Amygdala Brain diseaseUni-ZAP


De ression XR


50112 H othalamus Brain Uni-ZAP


282


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
XR


SO1 Anergic T-cell Anergic T-cell Cell Uni-ZAP
14 Line


XR


SO1 Bone marrow Bone marrow Bone marrow Uni-ZAP
16


XR


S0126Osteoblasts Osteoblasts Knee Cell Uni-ZAP
Line .


XR


50132Epithelial-TNFaAirway Epithelial Uni-ZAP
and INF


induced XR


50134Apoptotic T-cellapoptotic Cell Uni-ZAP
cells Line


XR


SO136PERM TF274 stromal cellBone marrowCell Lambda
Line


ZAP 11


50140eosinophil-ILS eosinophil lung Cell Uni-ZAP
induced Line


XR


50142Macrophage-oxLDLmacrophage- blood' Cell Uni-ZAP
Line


oxidized XR
LDL


treated


50144Macrophage (GM-CSFMacrophage Uni-ZAP
(GM-


treated) CSF treated) XR


50146prostate-editedprostate Prostate Uni-ZAP
BPH


XR


50148Normal ProstateProstate prostate Uni-ZAP


XR


SO150LNCAP prostate LNCAP Cell Prostate Cell Uni-ZAP
cell line Line Line


XR


50152PC3 Prostate PC3 prostate Uni-ZAP
cell line cell


line XR


50176Prostate, normal,Prostate prostate Uni-ZAP


subtraction XR
I


S0182Human B Cell Human B- Uni-ZAP
8866 Cell 8866


XR


S0192Synovial FibroblastsSynovial pSportl
Fibroblasts


control]


S0194S ovial h oxia Synovial S ortl
Fibroblasts


50196Synovial 1L-1/TNFSynovial pSportl
Fibroblasts


stimulated


501987TM-pbfd PBLS, 7TM PCRII


rece for
enriched


502027TM-pbdd PBLS, 7TM PCRII


rece for
enriched


50206Smooth Muscle- Smooth musclePulmanaryCell pBluescript
HASTE Line


normalized arte


50208Messan ial cell,Messan ial S ortl
frac I cell


S0210Messan ial cell,Messangial S ortl
frac 2 cell


50212Bone Marrow Bone Marrow pSportl
Stromal


Cell, untreatedStromal


Cell,untreated


S0214Human Osteoclastoma,Osteoclastomabone diseaseUni-ZAP
re-


excision XR


50216Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line


induced induced XR


50218Apoptotic T-cell,apoptotic Cell Uni-ZAP
re- cells Line


excision XR


50222H. Frontal H. Brain, Brain diseaseUni-ZAP
Frontal


cortex,epileptic;re-Cortex, Epileptic XR


excision


S0228PSMIX PBLS, 7TM PCRII


rece for
enriched


S0242Synovial FibroblastsSynovial pSportl
Fibroblasts


Ill/TNF , subt


50250Human OsteoblastsHuman OsteoblastsFemur diseasepCMVSport
II


2.0


283


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
502527TM-PIMIX PBLS, 7TM PCRII
rece for
enriched


50260Spinal Cord, Spinal cord spinal Uni-ZAP
re-excision cord XR


S0264PPMIX PPMIX (HumanPituitary PCRII
Pituitary)


S0268PRMIX PRMIX (Humanprostate PCRII
Prostate)


50270PTMIX PTMIX (HumanThymus PCR11
Th us)


50274PCMIX PCMIX (HumanBrain PCRII
Cerebellum)


50276Synovial hypoxia-RSFSynovial Synovial pSportl
subtracted fobroblasts tissue
(rheumatoid)


50278H Macrophage Macrophage Uni-ZAP
(GM-CSF (GM- XR
treated), re-excisionCSF treated)


S0280Human Adipose Human Adipose Uni-ZAP
Tissue, Tissue XR
re-excision


S0282Brain Frontal Brain frontalBrain Lambda
Cortex, re- cortex ZAP II
excision


50300Frontal lobe,dementia;re-Frontal LobeBrain Uni-ZAP
excision dementia/Alzheimer' XR
's


50308S leen/normal S teen normal S ortl


50314Human Human diseasepSportl
osteoarthritis;fractionosteoarthritic
1 cartila a


50316Human Normal Human Normal pSportl
Cartilage,FractionCartila a
I


50318Human Normal Human Normal pSportl
Cartilage Cartila a
Fraction II


50328Palate carcinomaPalate carcinomaUvula diseaseS ortl


S0330Palate normal Palate normalUvula S ortl


50332Pha x carcinomaPha x carcinomaH o ha S ortl
x


50338Human OsteoarthriticHuman diseasepSportl
Cartilage Fractionosteoarthritic
III cartila a


S0342Adipocytes;re-excisionHuman Adipocytes Uni-ZAP
from Osteoclastoma XR


S0344Macrophage-oxLDL;macrophage- blood Cell Uni-ZAP
re- oxidized Line XR
excision LDL
treated


50346Human Amygdala;re-Amygdala Uni-ZAP
excision XR


S0348Cheek CarcinomaCheek Carcinoma diseaseS ortl


S0352L x Carcinoma La x carcinoma diseaseS ortl


S0354Colon Normal Colon NormalColon S ortl
II


S0356Colon CarcinomaColon CarcinomaColon diseaseS ortl


50358Colon Normal Colon NormalColon S ortl
III


50360Colon Tumor Colon Tumor Colon diseaseS ortl
II


50362Human GastrocnemiusGastrocnemius pSportl
muscle


50364Human Quadrice Quadrice S ortl
s s muscle


50366Human Soleus Soleus Muscle S ortl


S0372La x carcinoma Larynx carcinoma diseaseS ortl
III


S0374Normal colon Normal colon S ortl


50376Colon Tumor Colon Tumor diseaseS ortl


50378Pancreas normalPancreas pSport
PCA4 Normal I
No PCA4 No


50380Pancreas Tumor Pancreas diseasepSportl
PCA4 Tu Tumor
PCA4 Tu


S0382La x carcinoma La x carcinoma diseaseS ortl
IV


S0384Tongue carcinomaTongue carcinoma diseasepSportl
~


284


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
50386 Human Whole Whole brain Brain ZAP
Brain, re- Ex ress
excision


50388 Human Human diseaseUni-ZAP
Hypothalamus,schizophreHypothalamus, XR
nia, re-excisionSchizo hrenia


50392 Salivary Gland Salivary pSportl
gland;
normal


S0400 Brain; normal Brain; normal S ortl


50402 Adrenal Gland,normalAdrenal gland; pSportl
normal


50404 Rectum normal Rectum, normal S ortl


S040G Rectum tumour Rectum tumour S ortl


50408 Colon, normal Colon, normal S ortl


50410 Colon, tumour Colon, tumour S ortl


S0412 Temporal cortex-Temporal diseaseOther
Alzheizmer; cortex,
subtracted alzheimer


50414 Hippocampus, Hippocampus, Other
Alzheimer Alzheimer
~~Subtracted Subtracted


S0418 CHME Cell Line;treatedCHME Cell pCMVSport
Line; 3.0
hrs treated


50420 CHME Cell CHME Cell pSportl
Line,untreated line,
untreatetd


S0422 Mo7e Cell Line Mo7e Cell pCMVSport
GM-CSF Line 3.0
treated ( 1 GM-CSF treated
ng/ml) (In ml)


50424 TF-1 Cell Line TF-1 Cell pSportl
GM-CSF Line
Treated GM-CSF Treated


50426 Monocyte activated;Monocyte-activatedblood Cell Uni-ZAP
re- Line XR
excision


50428 Neutrophils human neutrophilsblood Cell Uni-ZAP
control; re- Line XR
excision


50432 Sinus piniformisSinus piniformis pSportl
Tumour Tumour


S0434 Stomach Normal Stomach Normal diseaseS ortl


S0436 Stomach Tumour Stomach Tumour diseaseS ortl


S0440 Liver Tumour Liver Tumour S ortl
Met 5 Tu


S0442 Colon Normal Colon Normal S ortl


S0444 Colon Tumor Colon Tumour diseaseS ortl


S0446 Ton ue Tumour Ton ue Tumour S ortl


S0448 La x Normal La x Normal S ortl


50450 Larynx Tumour Larynx Tumour S ortl


50452 Th us Th us S ortl


S0458 Thyroid Normal Thyroid normal pSportl
(SDCA2
No)


50464 La x Normal La x Normal S ortl


S0470 Adenocarcinoma PYFD diseaseS ortl


S0474 Human blood Platelets Blood Other
platelets latelets


53012 Smooth Muscle Smooth musclePulmanaryCell pBluescript
Serum arte Line
Treated, Norm


53014 Smooth muscle, Smooth musclePulmanaryCell pBluescript
serum arte Line
induced,re-exc


56014 H. hypothalamus,HypothalamusBrain ZAP
frac A Ex ress


56016 H. Frontal Cortex,H. Brain, Brain diseaseUni-ZAP
E ile tic Frontal XR
Cortex, E
ile tic


S6026 Frontal Lobe, Frontal LobeBrain Uni-ZAP
Dementia dementia/Alzheimer' XR
's


S6028 Human Manic Human Manic Brain diseaseUni-ZAP
Depression de ression XR
Tissue tissue


T0002 Activated T-cellsActivated Blood Cell pBluescript
T-Cell, Line


285


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
PBL fraction SK-


T0003Human Fetal Human Fetal pBluescript
Lung Lung


SK-


T0004Human White Human White pBluescript
Fat Fat


SK-


T0006Human Pineal Human Pinneal pBluescript
Gland


Gland SK-


T0008Colorectal TumorColorectal diseasepBluescript
Tumor


SK-


T0010Human Infant Human Infant Other
Brain Brain


T0023Human PancreaticHuman Pancreatic diseasepBluescript


Carcinoma Carcinoma SK-


T0039HSA 172 Cells Human HSA172 pBluescript
cell


line SK-


T0040HSC172 cells SA172 Cells pBluescript


SK-


T0041Jurkat T-cell Jurkat T-cell pBluescript
Gl phase


SK-


T0042Jurkat T-Cell, Jurkat T-Cell pBluescript
S phase Line


SK-


T0048Human Aortic Human Aortic pBluescript


Endothelium Endothelium SK-


T0049Aorta endothelialAorta endothelial pBluescript
cells +


TNF-a cells SK-


T0060Human White Human White pBluescript
Adipose Fat


SK-


T0067Human Thyroid Human Thyroid pBluescript


SK-
,


T0069Human Uterus, Human Uterus, pBluescript
normal


normal SK-


T0071Human Bone MarrowHuman Bone pBluescript


Marrow SK-


T0082Human Adult Human Adult pBluescript
Retina Retina


SK-


T0109Human (HCC) pBluescript
cell line


liver (mouse) SK-
metastasis,


remake


TO110Human colon pBluescript
carcinoma ,


(HCC cell line, SK-
remake


T0114Human (Caco-2) pBluescript
cell line,


adenocarcinoma, SK-
colon,


remake


TO1 Human Colon pBluescript
I Carcinoma
S


(HCC cell line SK-


L0002Atrium cDNA
library


Human heart


L0005Clontech human
aorta


of A+ mRNA #6572)


L0021Human adult
(K.Okubo)


L0022Human adult
lung 3"


directed MboI
cDNA


L0040Human colon
mucosa


L0055Human rom eloc
a


L0065Liver He G2
cell line.


L0097Subtracted human
retinal


e ent a ithelium
RPE)


LO105Human aorta aorta
polyA+


TFu'iwara)


L0118Human fetal brain
brain S.


Meier-Ewert


L0142Human placenta placenta
cDNA


TFu~iwara


LO151Human testis testis
(C. De


286


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
Smet)


LO157 Human fetal brain
brain


(TFu' i wara)


L0163 Human heart heart
cDNA


(YNakamura)


L0193 Human osteosarcomaosteosarcoma OsA-CL


EGracia


L0352 Normalized infant BA, M
brain, 13-


Bento Soares derived


L0355 P, Human foetal Bluescript
Brain


Whole tissue


L0361 Stratagene ovary ovary Bluescript


#937217) SK


L0362 Stratagene ovarian Bluescript
cancer


#937219) SK-


L0366 Stratagene schizoschizophrenic Bluescript
brain brain


S11 S-I1 frontal SK-
lobe


L0367 ~ NCI CGAP Sch Schwannoma Bluescript
l tumor


S K-


L0369 NCI CGAP AAI adrenal adenomaadrenal Bluescript
gland


S K-


L0370 Johnston frontalpooled frontalbrain Bluescript
cortex lobe


SK-


L0372 NCI CGAP Co colon tumor colon Bluescript
12


S K-


L0373 NCI CGAP Coll tumor colon Bluescript


S K-


L0374 NCI CGAP Co2 tumor colon Bluescript
.


S K-


L0375 NCI CGAP_Kid6 kidney tumorkidney Bluescript


SK-


L0378 NCI CGAP Lul lung tumor lung Bluescript


S K-


L0381 NCI CGAP_HN4 squamous pharynx Bluescript
cell


carcinoma SK-


L0382 NCI CGAP_Pr25 epithelium prostate Bluescript
(cell line)


SK-


L0384 NCI CGAP Pr23 prostate prostate Bluescript
tumor


S K-


L0386 NCI CGAP_HN3 squamous tongue Bluescript
cell


carcinoma SK-
from base


ofton ue


L0387 NCI CGAP_GCBO germinal tonsil Bluescript
center B-


cells SK-


L0388 NCI CGAP_HN6 normal gingiva Bluescript
(cell


l ine from SK-


immortalized
kerati


L0389 NCI CGAP_HNS normal gingiva Bluescript
(cell


l ine from SK-
primary


keratinocyt


L0393 B, Human Liver 11
tissue


L0435 Infant brain, lafmid
LLNL array BA


of Dr. M. Soares
INIB


L0438 normalized infanttotal brain brain lafmid
brain BA


cDNA


L0439 Soares infant whole Lafmid
brain 1NIB brain BA


L0455 Human retina retina eye lambda
cDNA gtl0


randomly primed


sublibrary


L0456 Human retina retina eye lambda
cDNA gt 10


Tsp509I-cleaved


sublibra


L0462 WATM1 lambdagtll
~


287


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
L0465TEST1, Human lambda
adult


Testis tissue nm1149


L0469T, Human adult Lambda


Rhabdomyosarcoma Zap
cell-


line


L0471Human fetal Lambda
heart,


Lambda ZAP Express ZAP


Ex ress


L0483Human pancreatic Lambda
islet


ZAPII


L0485STRATAGENE Humanskeletal leg muscle Lambda
muscle


skeletal muscle ZAPII
cDNA


library, cat.
#936215.


L0499NCI CGAP HSC2 stem cell bone AMP1
34+/38+ marrow


L0509NCI_CGAP_Lu26 invasive lung pAMPI


adenocarcinoma


L0512NCI_CGAP_Ov36 borderline ovary pAMP
ovarian 1


carcinoma


L0513NCI CGAP Ov37 early stage ovary pAMP
papillary 1


serous carcinoma


L0517NCI CGAP Prl AMPIO


L0518NCI CGAP Pr2 AMP10


L0519NCI CGAP Pr3 AMP 10


L0520NCI_CGAP_Alvl alveolar pAMPlO


rhabdomyosarcoma
.


L0521NCI CLAP Ewl Ewin "s sarcoma AMP10


L0522NCI CGAP Kidl kidne AMP10


L0523NCI CGAP Li liposarcoma AMP10
2


L0526NCI CGAP Prl2 metastatic pAMPlO
prostate


b one lesion


L0527NCI CGAP Ov2 ova AMP10


L0528NCI CGAP Pr5 rostate AMP10


L0529NCI CGAP Pr6 prostate AMP10


L0534Chromosome 7 brain brain pAMPlO
Fetal


Brain cDNA Libra


L0535NCI CGAP Br5 infiltratingbreast pAMPlO
ductal


carcinoma


L0542NCI CGAP Prl normal prostaticprostate pAMPlO
l


a ithelial
cells


L0543NCI CGAP-Pr9 normal prostaticprostate pAMPlO


a ithelial
cells


L0544NCI CGAP Pr4 prostatic prostate pAMPlO


intraepithelial


neoplasia
- high


ade


L0545NCI CGAP Pr4.l prostatic prostate pAMPlO


intraepithelial


neoplasia
- high


ade


L0549NCI_CGAP_HN10 carcinoma pAMPlO
in situ


from retromolar


tri one


L0551NCI CGAP HN7 normal squamous pAMPlO


epithelium,
floor of


mouth


L0555NCI CLAP Lu34 lar a cell lun AMP10
carcinoma


L0557NCI CGAP Lu21 small cell lung AMP10
carcinoma


L0558NCI CGAP_Ov40 endometrioidovary pAMPlO


ovarian metastasis


L0564Jia bone marrowbone marrow Bluescri
stroma stroma t


L0565Normal Human Bone Hip pBluescript


Trabecular Bone
Cells


L0581Strata ene liver liver pBluescript
(#937224)


288


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
SK


L0586 HTCDL1 pBluescript


r
SK(-)


L0587 Stratagene colon pBluescript
HT29


(#937221 ) SK-


L0588 Stratagene endothelial pBluescript
cell


937223 SK-


L0589 Stratagene fetal pBluescript
retina


937202 SK-


L0590 Stratagene fibroblast pBluescript


(#937212)
SK-


L0591 Stratagene HeLa pBluescript
cell s3


937216 SK-


L0592 Stratagene hNT pBluescript
neuron


(#937233) SK-


L0593 Stratagene pBluescript


neuroepithelium SK-


" #937231)


L0594 Stratagene pBluescript


neuroepithelium SK-


NT2RAMI 937234


L0595 Stratagene NT2 neuroepithelialbrain pBluescript
neuronal cells


recursor 937230 SK-


L0596 Stratagene colon colon pBluescript


#937204) SK-


L0597 Stratagene corneal cornea pBluescript
stroma


(#937222)
SK-


L0598 Morton Fetal cochlea ear pBluescript
Cochlea ,


SK-


L0599 Stratagene lung lung pBluescript
(#937210)


SK-


L0600 Weizmann Olfactoryolfactory nose pBluescript
epithelium


E ithelium SK-


L0601 Stratagene pancreas pancreas pBluescript


#937208) SK-


L0602 Pancreatic Isletpancreatic pancreas pBluescript
islet


SK-


L0603 Stratagene placenta placenta pBluescript


(#937225)
SK-


L0604 Stratagene musclemuscle skeletal pBluescript
937209


muscle SK-


L0605 Stratagene fetalfetal spleenspleen pBluescript
spleen


(#937205)
SK-


L0606 NCI CGAP_LymS follicular lymph pBluescript
lymphoma node


SK-


L0607 NCI CGAP_Lym6 mantle cell lymph pBluescript
node


I homa SK-


L0608 Stratagene lunglung carcinomalung NCI-H69 pBluescript
carcinoma


937218 SK-


L0617 Chromosome 22 pBluescriptl
exon


IKS+


L0619 Chromosome 9 pBluescriptl
exon II


IKS+


L0622 HM1 pcDNAlI


Invitro
en


L0625 NCI_CGAP_AR1 bulk alveolar pCMV-
tumor


SPORT2


L0627 NCI_CGAP_Col bulk tumor colon pCMV-


SPORT2


L0628 NCI CGAP Ovl ovary bulk ovary pCMV-
tumor


S PORT2


L0629 NCI CGAP Mel3 metastatic bowel pCMV-
(skin


melanoma rima SPORT4
to bowel )


289


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
L0631NCI CLAP Br7 breast pCMV-


SPORT4


' NCI CLAP PNS1 dorsal root peripheral pCMV-
L0635 ganglion


nervous SPORT4


s stem


L0636NCI CGAP_Pitl four pooled brain pCMV-
pituitary


adenomas PORT6
S


L0637NCI CLAP Bm53 three pooledbrain pCMV-


meningiomas SPORT6


L0638NCI CGAP Btn35 tumor, 5 brain pCMV-
pooled (see


d escri lion) SPORT6


L0639NCI CGAP Brn52 tumor, 5 brain pCMV-
pooled (see


d escri lion) SPORT6


L0640NCI CGAP_Brl8 four pooled breast pCMV-
high-


grade tumors, SPORT6


including
two rima


L0641NCI CLAP Col7 juvenile colon pCMV-
granulosa


tumor SPORT6


L0642NCI CGAP Cola moderately colon pCMV-


d ifferentiatedS PORT6


adenocarcinoma


L0643NCI CGAP Col9 moderately colon pCMV-


d ifferentiatedS PORT6


adenocarcinoma


L0644NCI CGAP Co20 moderately colon pCMV-


d ifferentiatedS PORT6


adenocarcinoma


L0645NCI CLAP Co21 moderately colon pCMV-


d ifferentiated SPORT6


adenocarcinoma


L0646NCI CGAP Col4 moderately- colon pCMV-


d ifferentiatedS PORT6


adenocarcinoma


L0647NCI CGAP_Sar4 five pooled connective pCMV-


sarcomas, tissue SPORT6
including


m oid Ii
osarcoma


L0648NCI CGAP_Eso2 squamous esophagus pCM V-
cell


carcinoma SPORT6


L0649NCI CGAP_GUl 2 pooled genitourinary pCMV-
high-grade


transitionaltract SPORT6
cell


tumors


L0650NCI CGAP Kidl3 2 pooled kidney pCMV-
Wilms'


tumors, one S PORT6
primary


and one metast


L0651NCI CGAP_Kid8 renal cell kidney pCMV-
tumor


SPORT6


L0653NCI CLAP Lu28 two pooled lung pCMV-


squamous SPORT6
cell


carcinomas


L0655NCI CGAP-Lyml2 lymphoma, lymph pCMV-
node


f ollicular SPORT6
mixed


small and
lar a cell


L0656NCI CGAP_Ov38 normal epitheliumovary pCMV-


SPORT6


L0657NCI CGAP Ov23 tumor, 5 ovary pCMV-
pooled (see


d escri tion SPORT6


L0658NCI CGAP_Ov35 tumor, 5 ovary pCMV-
pooled (see


descri tion SPORT6


L0659NCI CGAP_Panl adenocarcinomapancreas pCMV-


S PORT6


L0661NCI CGAP Me115 malignant skin pCMV-


melanoma, S PORT6


metastatic
to lymph


node


290


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WO 01/55163 PCT/USO1/01358
LOGG2NCI CGAP Gas4 poorly differentiatedstomach pCMV-


adenocarcinoma SPORTG


with si net
r


LOG63NCI moderately- uterus pCMV-
CGAP
Ut2


_ differentiated SPORT6
_


endometrial


adenocarcino


LOGG4NCI CGAP poorly-differentiateduterus pCMV-
Ut3


_ endometrial SPORTG


adenocarcinoma,


LOGGSNCI CGAP Ut4 serous papillaryuterus pCMV-


carcinoma, PORT6
high S


rade, 2 ooled
t


LOGGGNCI well-differentiateduterus pCMV-
CGAP
Utl


_ endometrial SPORT6
_


adenocarcinoma,
7


L0667NCI CGAP CMLI myeloid cells,whole pCMV-
18 blood


C ML cases, SPORT6
pooled


BCR/ABL rearra


LOG8GStanley Frontalfrontal lobebrain pCR2.1-
SN pool 2 (see


description) TOPO


(Invitro
en)


LOG95Human Glialblastoma Brain BT-325 PCRII,
Cell


Invitro
en


L0G98Testis 2 PGEM


Szf(+)


L0717Gessler Wilms SPORTI
tumor


L0720PN001-Normal prostate pSportl
Human


Prostate


L0731Soares-pregnant uterus pT7T3-Pac
uterus-


NbHPU


L0740Soares melanocytemelanocyte pT7T3D


2NbH M (Pharmacia)


with
a


modified


of linker


L0741Soares adult brain pT7T3D
brain


N2b4HB55Y (Pharmacia)


with
a


modified


of linker


L0742Soares adult brain pT7T3D
brain


N2b5HB55Y (Pharmacia)


with
a


modified


of linker


L0743Soares breast breast pT7T3D
2NbHBst


(Pharmacia)


with
a


modified


of linker


L0744Soares breast breast pT7T3D
3NbHBst


(Pharmacia)


with
a


modified


of linker


L0745Soares retina retina eye pT7T3D
N2b4HR


(Pharmacia)


with
a


modified


of linker


L074GSoares retina retina eye pT7T3D
N2b5HR


(Pharmacia)


with
a


291


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
modified


olylinker


L0747heart_NbHH heart pT7T3D
Soares
fetal


_ (Pharmacia)
_
I ~~r


with
a


modified


olylinker


L0748Soares fetal Liver pT7T3D
liver spleen and


1 NFLS Spleen (Pharmacia)


with
a


modified


olylinker


L0749liver spleen Liver pT7T3D
Soares and
fetal


_ Spleen (Pharmacia)
_
1NFLS S1


- with
a


modified


of linker


L0750lung NbHLI lung pT7T3D
wSoares fetal


_ (Pharmacia)
9W -


with
a


modified


of linker


L0751Soares ovary ovarian tumorovary pT7T3D
tumor


NbHOT (Pharmacia)


with
a


modified


of linker


L0752parathyroid parathyroid parathyroid pT7T3D
tumor tumor
Soares


- gland (Pharmacia)'
NbHPA


with
a


modified


of linker


L0753gland N3H pineal pT7T3D
pineal gland
Soares


- (Pharmacia)
-
pC,


with
a


modified


of linker


L0754Soares placenta placenta pT7T3D
Nb2HP


(Pharmacia)


with
a


modified


olylinker


L0755placenta 8to9wee placenta pT7T3D
Soares


- (Pharmacia)
ks 2NbHP8to9W


- with
a


modified


of linker


L0756multiple sclerosismultiple pT7T3D
Soares sclerosis


_ lesions (Pharmacia)
2NbHMSP


with
a


modified


polylinker


V TYPE


L0757fibrobla senescent pT7T3D
Soares senescentfibroblast


_ (Pharmacia)
NbHSF
sts


_ with
a


modified


polylinker


V TYPE


L0758testis pT7T3D-
NHT
Soares


_ Pac
_


(Pharmacia)


with
a


modified


olylinker


292


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
L0759 Soares_total_fetus pT7T3D-
Nb2H


_ Pac
9w
F8


_ (Pharmacia)


with
a


modified


of linker


L0760 Barstead aorta aorta pT7T3D-
HPLRB3


Pac


(Pharmacia)


with
a


modified


olylinker


L0761 CGAP B-cell, chronic pT7T3D-
NCI
CLLI


_ lymphotic Pac
_ leukemia


(Pharmacia)


with
a


modified


olylinker


L0762 Brl.l breast pT7T3D-
NCI
CGAP


_ Pac
_


(Pharmacia)


with
a


modified


of linker


L0763 NCI breast pT7T3D-
CGAP
Br2


_ Pac
_


(Pharmacia)


with
a


modified


olylinker


L0764 NCI colon pT7T3D-
CGAP
Co3


_ Pac
_


(Pharmacia)


with
a


modified


of linker


L0765 NCI colon pT7T3D-
CGAP
Co4


_ Pac
_


(Pharmacia)


with
a


modified


of linker


L0766 NCI CGAP germinal pT7T3D-
GCB1 center B


_ cell
Pac


(Pharmacia)


with
a


modified


of linker


L0767 NCI CGAP_GC3 pooled germ pT7T3D-
cell


tumors Pac


(Pharmacia)


with
a


modified


olylinker


L0768 GC4 pooled germ pT7T3D-
NCI CGAP cell


_ tumors Pac
~


(Pharmacia)


with
a


modified


of linker


L0769 Brn25 anaplastic brain pT7T3D-
NCI CGAP


_ oligodendroglioma Pac


(Pharmacia)


with
a


modified


293


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
of linker


L0770 NCI CLAP Brn23 glioblastomabrain pT7T3D-


(pooled) Pac


(Pharmacia)


with
a


modified


of linker


L0771 NCI_CGAP_Co8 adenocarcinomacolon pT7T3D-


Pac


(Pharmacia)


with
a


modified


of linker


L0772 CGAP_CoIO colon tumor colon pT7T3D-
NCI RER+


_ Pac


(Pharmacia)


with
a


modified


of linker


L0773 NCI_CGAP_Co9 colon tumor colon pT7T3D-
RER+


Pac


. (Pharmacia)


with
a


modified


olylinker


L0774 NCI_CGAP_Kid3 kidney pT7T3D-


Pac


(Pharmacia)


with
a


modified


of linker


L0775 NCI CGAP_Kids 2 pooled kidney pT7T3D-
tumors


( clear cell Pac
type)


(Pharmacia)


with
a


modified


of linker


L0776 CGAP_Lu5 carcinoid lung pT7T3D-
NCI


_ Pac


(Pharmacia)


with
a


modified


of linker


L0777 Soares_NhHMPu_S1Pooled humanmixed pT7T3D-
(see


melanocyte, below) Pac
fetal


heart, and (Pharmacia)
pregnant


with
a


modified


of linker


L0779 NFL_T_GBC_S1 pooled pT7T3D-
Soares


_ Pac


(Pharmacia)


with
a


modified


of linker


L0780 Soares_NSF F8 pooled pT7T3D-
9W OT


PA Pac
P
S I


_ (Pharmacia)
_


with
a


modified


of linker


L0783 NCI CGAP Pr22 normal prostateprostate pT7T3D-


P ac


(Pharmacia)


with
a


294


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
modified


olylinker


L0784 NCI CGAP leiomyosarcomasoft pT7T3D-
Lei2 tissue


- Pac


(Pharmacia)


with
a


modified


olylinker


L0785 Barstead spleen spleen pT7T3D-
HPLRB2


Pac


(Pharmacia)


with
a


modified


of linker


L0786 Soares_NbHFB whole pT7T3D-
brain


Pac


(Pharmacia)


with
a


modified


olylinker


L0787 NCI_CGAP_Subl pT7T3D-


Pac


(Phannacia)


with
a


modified


of linker


L0788 NCI pT7T3D-
CGAP_Sub2


_ Pac


(Pharmacia)


with
a


modified


of linker


L0789 NCI_CGAP_Sub3 pT7T3D-


Pac


(Pharmacia)


with
a


modified


of linker


L0790 NCI_CGAP_Sub4 pT7T3D-


Pac


(Pharmacia)


with
a


modified


of linker


L0791 NCI_CGAP pT7T3D-
SubS


_ Pac


(Pharmacia)


with
a


modified


of linker


L0792 NCI_CGAP pT7T3D-
Sub6


_ Pac


(Pharmacia)


with
a


modified


of linker


L0794 NCI CGAP_GC6 pooled germ pT7T3D-
cell


tumors Pac


(Pharmacia)


with
a


modified


of linker


L0796 NCI_CGAP_Bm50 medulloblastomabrain pTTT3D-


Pac


(Pharmacia)


295


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
with
a


modified


olylinker


L0800 CGAP_Col6 colon tumor,colon pT7T3D-
NCI RER+


_ Pac


(Pharmacia)


with
a


modified


of linker


L0803 CGAP_Kidll kidney pT7T3D-
NCI


_ Pac


(Pharmacia)


with
a


modified


of linker


L0804 Kid 12 2 pooled kidney pT7T3D-
NCI CGAP tumors


_ clear cell , Pac
( type)


(Pharmacia)


with
a


modified


polylinker


L0805 Lu24 carcinoid lung pT7T3D-
NCI
CGAP


_ Pac
_


(Pharmacia)


with
a


modified


of linker


L080G Lul9 squamous lung pT7T3D-
NCI CLAP cell


- carcinoma, Pac
poorly


differentiated (Pharmacia)
(4


with
a


modified


of linker


L0808 Barstead prostate prostate pT7T3D-
BPH


HPLRB4 1 Pac


(Pharmacia)


with
a


- modified


olylinker


L0809 NCI prostate pT7T3D-
CGAP
Pr28


_ Pac
_


(Pharmacia)


with
a


modified


olylinker


L2250 Human cerebral cerebral
cortex cortex


TABLE 5
OMIM Description
Reference


100730 Myasthenia gravis, neonatal transient


106165 Hypertension, essential, 145500


107280 Cerebrovascular disease, occlusive


107280 Alpha-1-antichymot sin deficiency


107400 Emphysema


107400 Emphysema-cirrhosis


117700 ~ [Hypoceruloplasminemia, hereditary]


296


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
117700 Hemosiderosis, systemic, due to aceruloplasminemia


118800 Choreoathetosis, familial paroxysmal


121050 Contractural arachnodactyly, congenital


122500 [Transcortin deficiency]


123660 Cataract, Coppock-like


129490 Ectodermal dysplasia-3, anhidrotic


131400 Eosinophilia, familial


135600 Ehlers-Danlos syndrome, type X


138040 Cortisol resistance


150210 Lactoferrin-deficient neutrophils, 245480


153455 Cutis laxa, recessive, type I, 219100


154705 Marfan syndrome, type II


157655. Lactic acidosis due to defect in iron-sulfur cluster
of complex I


159000 Muscular dystrophy, limb-girdle, type 1A


169600 Hailey-Hailey disease


179095 Male infertility


180380 Night blindness, congenital stationery, rhodopsin-related


180380 Retinitis pigmentosa, autosomal recessive


180380 Retinitis pigmentosa-4, autosomal dominant


181460 Schistosoma mansoni, susce tibility/resistance
to


186860 Leukemia/lymphoma, T-cell


186960 Leukemiallymphoma, T-cell


190000 Atransfernnemia


192974 Neonatal alloimmune thrombocytopenia


192974 Glyco rotein Ia deficiency


193300 Renal cell carcinoma


193300 von Hippel-Lindau syndrome


201460 Acyl-CoA dehydrogenase, long chain, deficiency
of


203500 Alkaptonuria


205100 Amyotrophic lateral sclerosis, juvenile


213700 Cerebrotendinous xanthomatosis


222900 Sucrose intolerance


227646 Fanconi anemia, type D


232050 Propionicacidemia, t a II or pccB t a


245200 Krabbe disease


253260 Biotinidase deficiency


262000 Bjornstad syndrome


276902 Usher syndrome, t a 3


278720 Xeroderma pigmentosum, grou C


300047 Mental retardation, X-linked 20


300062 Mental retardation, X-linked 14


300600 Ocular albinism, Forsius-Eriksson t a


309470 Mental retardation, X-linked, syndromic-3, with
spastic diplegia


309500 Renpenning syndrome-1


309610 Mental retardation, X-linked, syndromic-2, with
dysmorphism and
cerebral atrophy


310500 ~ Night blindness, congenital stationary, type
1


297


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
310600 Norrie disease


310600 Exudative vitreoretinopathy, X-linked, 305390


311050 Optic atrophy, X-linked


312060 Properdin deficiency, X-linked


600258 Colorectal cancer, hereditary non olyposis, type
3


600334 Tibial muscular dystrophy


600807 Bronchial asthma


600882 Charcot-Marie-Tooth neuropathy-2B


601154 Cardiomyopathy, dilated, 1 E


601199 Neonatal hyperparathyroidism, 239200


601199 Hypocalcemia, autosomal dominant, 601198


601199 Hypocalciuric hypercalcemia, type I, 145980


601253. Muscular dystrophy, limb-girdle, t a IC


601277 Ichthyosis, lamellar, type 2


601318 Diabetes mellitus, insulin-de endent, 13


601471 Moebius syndrome-2


601596 Charcot-Marie-Tooth neuropathy, demyelinating


601682 Glaucoma 1 C, primary open angle


601692 Reis-Bucklers corneal dystrophy


601692 Corneal dystrophy, Avellino type


601692 Corneal dystro hy, Groenouw t a I, 121900


601692 Corneal dystrophy, lattice t a I, 122200


601841 Protein C inhibitor deficiency


602011 Pancreatic endocrine tumors


602089 Hemangioma, capillary, hereditary


602121 Deafness, autosomal dominant nonsyndromic sensorineural,
l, 124900


602460 Deafness, autosomal dominant 15, 602459


Polynucleotide and Polypeptide Variants
[85] The present invention is directed to variants of the polynucleotide
sequence
disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide
sequences
encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID
NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A,
nucleotide sequences
encoding the polypeptide as defined in column 7 of Table 1A, the nucleotide
sequence as
defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the
polypeptide
encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2,
the nucleotide
sequence as defined in column 6 of Table 1 B, nucleotide sequences encoding
the polypeptide
encoded by the nucleotide sequence as defined in column 6 of Table 1B, the
cDNA sequence
298


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
contained in Clone >D NO:Z, and/or nucleotide sequences encoding the
polypeptide encoded
by the cDNA sequence contained in Clone ID NO:Z.
[86] The present invention also encompasses variants of the polypeptide
sequence
disclosed in SEQ >D NO:Y, the polypeptide sequence as defined in column 7 of
Table 1A, a
polypeptide sequence encoded by the polynucleotide sequence in SEQ m NO:X, a
polypeptide sequence encoded by the nucleotide sequence as defined in columns
8 and 9 of
Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined
in column 6
of Table IB, a polypeptide sequence encoded by the complement of the
polynucleotide
sequence in SEQ >D NO:X, and/or a polypeptide sequence encoded by the cDNA
sequence
contained in Clone ID NO:Z.
[87] "Variant" refers to a polynucleotide or polypeptide differing from the
polynucleotide or polypeptide of the present invention, but retaining
essential properties
thereof. Generally, variants are overall closely similar, and, in many
regions, identical to the
polynucleotide or polypeptide of the present invention.
[88] Thus, one aspect of the invention provides an isolated nucleic acid
molecule
comprising, or alternatively consisting of, a polynucleotide having a
nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence described in
SEQ >Z7 NO:X
or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence
in SEQ >D
NO:X or the cDNA in Clone 1T7 NO:Z which encodes the complete amino acid
sequence of
SEQ >D NO:Y or the complete amino acid sequence encoded by the cDNA in Clone
ID
NO:Z; (c) a nucleotide sequence in SEQ >D NO:X or the cDNA in Clone >D NO:Z
which
encodes a mature polypeptide; (d) a nucleotide sequence in SEQ m NO:X or the
cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a
polypeptide;
(e) a nucleotide sequence in SEQ m NO:X or the cDNA sequence of Clone m NO:Z,
which
encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence
encoding a
polypeptide comprising the complete amino acid sequence of SEQ >D NO:Y or the
complete
amino acid sequence encoded by the cDNA in Clone >D NO:Z; (g) a nucleotide
sequence
encoding a mature polypeptide of the amino acid sequence of SEQ >D NO:Y or the
amino
acid sequence encoded by the cDNA in Clone >D NO:Z; (h) a nucleotide sequence
encoding
a biologically active fragment of a polypeptide having the complete amino acid
sequence of
SEQ >D NO:Y or the complete amino acid sequence encoded by the cDNA in Clone
>D
NO:Z; (i) a nucleotide sequence encoding an antigenic fragment of a
polypeptide having the
complete amino acid sequence of SEQ 1D NO:Y or the complete amino acid
sequence
299


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
encoded by the cDNA in Clone ID NO:Z; and (j) a nucleotide sequence
complementary to
any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or
(i) above.
[89] The present invention is also directed to nucleic acid molecules which
comprise, or
alternatively consist of, a nucleotide sequence which is at least 80%, 85%,
90%, 95%, 96%,
97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide
sequences in (a),
(b), (c), (d), (e), (f), (g), (h), (i), or (j) above, the nucleotide coding
sequence in SEQ m
NO:X or the complementary strand thereto, the nucleotide coding sequence of
the cDNA
contained in Clone >D NO:Z or the complementary strand thereto, a nucleotide
sequence
encoding the polypeptide of SEQ m NO:Y, a nucleotide sequence encoding a
polypeptide
sequence encoded by the nucleotide sequence in SEQ >D NO:X, a polypeptide
sequence
encoded by the complement of the polynucleotide sequence in SEQ >D NO:X,. a
nucleotide
sequence encoding the polypeptide encoded by the cDNA contained in Clone m
NO:Z, the
nucleotide coding sequence in SEQ )D NO:X as defined in columns 8 and 9 of
Table 2 or the
complementary strand thereto, a nucleotide sequence encoding the polypeptide
encoded by
the nucleotide sequence in SEQ >D NO:X as defined in columns 8 and 9 of Table
2 or the
complementary strand thereto, the nucleotide coding sequence in SEQ m NO:B as
defined in
column 6 of Table 1 B or the complementary strand thereto, a nucleotide
sequence encoding
the polypeptide encoded by the nucleotide sequence in SEQ m NO:B as defined in
column 6
of Table 1 B or the complementary strand thereto, the nucleotide sequence in
SEQ >D NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A or the
complementary
strand thereto, nucleotide sequences encoding the polypeptide as defined in
column 7 of
Table 1 A or the complementary strand thereto, and/or polynucleotide fragments
of any of
these nucleic acid molecules (e.g., those fragments described herein).,
Polynucleotides which
hybridize to the complement of these nucleic acid molecules under stringent
hybridization
conditions or alternatively, under lower stringency conditions, are also
encompassed by the
invention, as are polypeptides encoded by these polynucleotides and nucleic
acids.
(90) In a preferred embodiment, the invention encompasses nucleic acid
molecules
which comprise, or alternatively, consist of a polynucleotide which hybridizes
under
stringent hybridization conditions, or alternatively, under lower stringency
conditions, to a
polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as
are polypeptides encoded
by these polynucleotides. In another preferred embodiment, polynucleotides
which hybridize
to the complement of these nucleic acid molecules under stringent
hybridization conditions,
300


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
or alternatively, under lower stringency conditions, are also encompassed by
the invention, as
ire polypeptides encoded by these polynucleotides.
[91 ] In another embodiment, the invention provides a purified protein
comprising, or
alternatively consisting of, a polypeptide having an amino acid sequence
selected from the
group consisting o~ (a) the complete amino acid sequence of SEQ >D NO:Y or the
complete
amino acid sequence encoded by the cDNA in Clone >D NO:Z; (b) the amino acid
sequence
of a mature form of a polypeptide having the amino acid sequence of SEQ >Z7
NO:Y or the
amino acid sequence encoded by the cDNA in Clone >D NO:Z; (c) the amino acid
sequence
of a biologically active fragment of a polypeptide having the complete amino
acid sequence
of SEQ >D NO:Y or the complete amino acid sequence encoded by the cDNA in
Clone )D
NO:Z; and (d) the amino acid sequence of an antigenic fragment of a
polypeptide having the
complete amino acid sequence of SEQ >D NO:Y or the complete amino acid
sequence
encoded by the cDNA in Clone m NO:Z.
[92] The present invention is also directed to proteins which comprise, or
alternatively
consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%,
99% or 100%, identical to, for example, any of the amino acid sequences in
(a), (b), (c), or
(d), above, the amino acid sequence shown in SEQ >D NO:Y, the amino acid
sequence
encoded by the cDNA contained in Clone ~ NO:Z, the amino acid sequence of the
polypeptide encoded by the nucleotide sequence in SEQ m NO:X as defined in
columns 8
and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the
nucleotide
sequence in SEQ B7 NO:B as defined in column 6 of Table 1B, the amino acid
sequence as
defined in column 7 of Table 1 A, an amino acid sequence encoded by the
nucleotide
sequence in SEQ 1D NO:X, and an amino acid sequence encoded by the complement
of the
polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are
also
provided (e.g., those fragments described herein). Further proteins encoded by
polynucleotides which hybridize to the complement of the nucleic acid
molecules encoding
these amino acid sequences under stringent hybridization conditions or
alternatively, under
lower stringency conditions, are also encompassed by the invention, as are the
polynucleotides encoding these proteins.
[93] By a nucleic acid having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended that the
nucleotide sequence of the nucleic acid is identical to the reference sequence
except that the
nucleotide sequence may include up to five point mutations per each 100
nucleotides of the
301


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
reference nucleotide sequence encoding the polypeptide. In other words, to
obtain a nucleic
acid having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence,
up to 5% of the nucleotides in the reference sequence may be deleted or
substituted with
another nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the
reference sequence may be inserted into the reference sequence. The query
sequence may be
an entire sequence referred to in Table 1A or 2 as the ORF (open reading
frame), or any
fragment specified as described herein.
[94J As a practical matter, whether any particular nucleic acid molecule or
polypeptide
is at least 80%, 85%, 90%. 95%, 96%, 97%, 98% or 99% identical to a nucleotide
sequence
of the present invention can be determined conventionally using known computer
programs.
A preferred method for determining the best overall match between a query
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, can be determined using the FASTDB computer program based
on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a
sequence alignment
the query and subject sequences are both DNA sequences. An RNA sequence can be
compared by converting U's to T's. The result of said global sequence
alignment is
expressed as percent identity. Preferred parameters used in a FASTDB alignment
of DNA
sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4,
Mismatch Penalty=l,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=S, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide
sequence,
whichever is shorter.
[95) If the subject sequence is shorter than the query sequence because of 5'
or 3'
deletions, not because of internal deletions, a manual correction must be made
to the results.
This is because the FASTDB program does not account for 5' and 3' truncations
of the
subject sequence when calculating percent identity. For subject sequences
truncated at the 5'
or 3' ends, relative to the query sequence, the percent identity is corrected
by calculating the
number of bases of the query sequence that are S' and 3' of the subject
sequence, which are
not matched/aligned, as a percent of the total bases of the query sequence.
Whether a
nucleotide is matched/aligned is determined by results of the FASTDB sequence
alignment.
This percentage is then subtracted from the percent identity, calculated by
the above
FASTDB program using the specified parameters, to arrive at a final percent
identity score.
This corrected score is what is used for the purposes of the present
invention. Only bases
outside the 5' and 3' bases of the subject sequence, as displayed by the
FASTDB alignment,
302


CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
which are not matched/aligned with the query sequence, are calculated for the
purposes of
manually adjusting the percent identity score.
[96] For example, a 90 base subject sequence is aligned to a 100 base query
sequence to
determine percent identity. The deletions occur at the S' end of the subject
sequence and
therefore, the FASTDB alignment does not show a matched/alignment of the first
10 bases at
5' end. The 10 unpaired bases represent 10% of the sequence (number of bases
at the S' and
3' ends not matched/total number of bases in the query sequence) so 10% is
subtracted from
the percent identity score calculated by the FASTDB program. If the remaining
90 bases
were perfectly matched the final percent identity would be 90%. In another
example, a 90
base subject sequence is compared with a 100 base query sequence. This time
the deletions
are internal deletions so that there are no bases on the 5' or 3' of the
subject sequence which
are not matched/aligned with the query. In this case the percent identity
calculated by
FASTDB is not manually corrected. Once again, only bases 5' and 3' of the
subject
sequence which are not matched/aligned with the query sequence are manually
corrected for.
No other manual corrections are to be made for the purposes of the present
invention. .
(97] By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that the
amino acid sequence of the subject polypeptide is identical to the query
sequence except that
the subject polypeptide sequence may include up to five amino acid alterations
per each 100
amino acids of the query amino acid sequence. In other words, to obtain a
polypeptide
having an amino acid sequence at least 95% identical to a query amino acid
sequence, up to
S% of the amino acid residues in the subject sequence may be inserted,
deleted, (indels) or
substituted with another amino acid. These alterations of the reference
sequence may occur
at the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere
between those terminal positions, interspersed either individually among
residues in the
reference sequence or in one or more contiguous groups within the reference
sequence.
[98] As a practical matter, whether any particular polypeptide is at least
80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid
sequence of a
polypeptide referred to in Table 1A (e.g., the amino acid sequence identified
in column 6) or
Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the
polynucleotide
sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the
amino acid
sequence of the polypeptide encoded by the polynucleotide sequence in SEQ >D
NO:B as
defined in column 6 of Table 1B or a fragment thereof, the amino acid sequence
of the
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polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment
thereof, or
the amino acid sequence of the polypeptide encoded by cDNA contained in Clone
ID NO:Z,
or a fragment thereof, can be determined conventionally using known computer
programs. A
preferred method for determining the best overall match between a query
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, can be determined using the FASTDB computer program based
on the
algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a
sequence alignment
the query and subject sequences are either both nucleotide sequences or both
amino acid
sequences. The result of said global sequence alignment is expressed as
percent identity.
Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,
k-
tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0,
Cutoff
Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
[99] If the subject sequence is shorter than the query sequence due to N- or C-
terminal
deletions, not because of internal deletions, a manual correction must be made
to the results.
This is because the FASTDB program does not account for N- and C-terminal
truncations of
the subject sequence when calculating global percent identity. For subject
sequences
truncated at the N- and C-termini, relative to the query sequence, the percent
identity is
corrected by calculating the number of residues of the query sequence that are
N- and C-
terminal of the subject sequence, which are not matched/aligned with a
corresponding subject
residue, as a percent of the total bases of the query sequence. Whether a
residue is
matched/aligned is determined by results of the FASTDB sequence alignment.
This
percentage is then subtracted from the percent identity, calculated by the
above FASTDB
program using the specified parameters, to arrive at a final percent identity
score. This final
percent identity score is what is used for the purposes of the present
invention. Only residues
to the N- and C-termini of the subject sequence, which are not matched/aligned
with the
query sequence, are considered for the purposes of manually adjusting the
percent identity
score. That is, only query residue positions outside the farthest N- and C-
terminal residues
of the subject sequence.
[100] For example, a 90 amino acid residue subject sequence is aligned with a
100
residue query sequence to determine percent identity. The deletion occurs at
the N-terminus
of the subject sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired
residues
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represent 10% of the sequence (number of residues at the N- and C- termini not
matched/total
number of residues in the query sequence) so 10% is subtracted from the
percent identity
score calculated by the FASTDB program. If the remaining 90 residues were
perfectly
matched the final percent identity would be 90%. In another example, a 90
residue subject
sequence is compared with a 100 residue query sequence. This time the
deletions are internal
deletions so there are no residues at the N- or C-termini of the subject
sequence which are not
matched/aligned with the query. In this case the percent identity calculated
by FASTDB is
not manually corrected. Once again, only residue positions outside the N- and
C-terminal
ends of the subject sequence, as displayed in the FASTDB alignment, which are
not
matched/aligned with the query sequnce are manually corrected for. No other
manual
corrections are to made for the purposes of the present invention.
[101] The polynucleotide variants of the invention may contain alterations in
the coding
regions, non-coding regions, or both. Especially preferred are polynucleotide
variants
containing alterations which produce silent substitutions, additions, or
deletions, but do not
alter the properties or activities of the encoded polypeptide. Nucleotide
variants produced by
silent substitutions due to the degeneracy of the genetic code are preferred.
Moreover,
polypeptide variants in which less than 50, less than 40, less than 30, less
than 20, less than
10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or
added in any
combination are also preferred. Polynucleotide variants can be produced for a
variety of
reasons, e.g., to optimize codon expression for a particular host (change
codons in the human
mRNA to those preferred by a bacterial host such as E. coli).
[102] Naturally occurring variants are called "allelic variants," and refer to
one of several
alternate forms of a gene occupying a given locus on a chromosome of an
organism. (Genes
II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic
variants can vary at
either the polynucleotide and/or polypeptide level and are included in the
present invention.
Alternatively, non-naturally occurring variants may be produced by mutagenesis
techniques
or by direct synthesis.
[103] Using known methods of protein engineering and recombinant DNA
technology,
variants may be generated to improve or alter the characteristics of the
polypeptides of the
present invention. For instance, one or more amino acids can be deleted from
the N-terminus
or C-terminus of the polypeptide of the present invention without substantial
loss of
biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988
(1993))
reported variant KGF proteins having heparin binding activity even after
deleting 3, 8, or 27
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amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up
to ten times
higher activity after deleting 8-10 amino acid residues from the carboxy
terminus of this
protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
[104] Moreover, ample evidence demonstrates that variants often retain a
biological
activity similar to that of the naturally occurring protein. For example,
Gayle and coworkers
(J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational
analysis of human
cytokine IL-la. They used random mutagenesis to generate over 3,500 individual
IL-la
mutants that averaged 2.5 amino acid changes per variant over the entire
length of the
molecule. Multiple mutations were examined at every possible amino acid
position. The
investigators found that "[m]ost of the molecule could be altered with little
effect on either
[binding or biological activity]." In fact, only 23 unique amino acid
sequences, out of more
than 3,500 nucleotide sequences examined, produced a protein that
significantly differed in
activity from wild-type.
[105 Furthermore, even if deleting one or more amino acids from the N-terminus
or C-
terminus of a polypeptide results in modification or loss of one or more
biological functions,
other biological activities may still be retained. For example, the ability of
a deletion variant
to induce and/or to bind antibodies which recognize the secreted form will
likely be retained
when less than the majority of the residues of the secreted form are removed
from the N-
terminus or C-terminus. Whether a particular polypeptide lacking N- or C-
terminal residues
of a protein retains such immunogenic activities can readily be determined by
routine
methods described herein and otherwise known in the art.
[106] Thus, the invention further includes polypeptide variants which show a
functional
activity (e.g., biological activity) of the polypeptides of the invention.
Such variants include
deletions, insertions, inversions, repeats, and substitutions selected
according to general rules
known in the art so as have little effect on activity.
[107J The present application is directed to nucleic acid molecules at least
80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences
disclosed
herein, (e.g., encoding a polypeptide having the amino acid sequence of an N
and/or C
terminal deletion), irrespective of whether they encode a polypeptide having
functional
activity. This is because even where a particular nucleic acid molecule does
not encode a
polypeptide having functional activity, one of skill in the art would still
know how to use the
nucleic acid molecule, for instance, as a hybridization probe or a polymerase
chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present invention that
do not encode
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a polypeptide having functional activity include, inter alia, (1) isolating a
gene or allelic or
splice variants thereof in a cDNA library; (2) in situ hybridization (e.g.,
"FISH") to
metaphase chromosomal spreads to provide precise chromosomal location of the
gene, as
described in Verma et al., Human Chromosomes: A Manual of Basic Techniques,
Pergamon
Press, New York (1988); (3) Northern Blot analysis for detecting mRNA
expression in
specific tissues (e.g., normal or diseased tissues); and (4) in situ
hybridization (e.g.,
histochemistry) for detecting mRNA expression in specific tissues (e.g.,
normal or diseased
tissues).
[108] Preferred, however, are nucleic acid molecules having sequences at least
80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid
sequences
disclosed herein, which do, in fact, encode a polypeptide having functional
activity. By a
polypeptide having "functional activity" is meant, a polypeptide capable of
displaying one or
more known functional activities associated with a full-length (complete)
protein of the
invention. Such functional activities include, but are not limited to,
biological activity,
antigenicity [ability to bind (or compete with a polypeptide of the invention
for binding) to
an anti-polypeptide of the invention antibody], immunogenicity (ability to
generate antibody
which binds to a specific polypeptide of the invention), ability to form
multimers with
polypeptides of the invention, and ability to bind to a receptor or ligand for
a polypeptide of
the invention.
[109] The functional activity of the polypeptides, and fragments, variants and
derivatives
of the invention, can be assayed by various methods.
[110] For example, in one embodiment where one is assaying for the ability to
bind or
compete with a full-length polypeptide of the present invention for binding to
an anti-
polypetide antibody; various immunoassays known in the art can be used,
including but not
limited to, competitive and non-competitive assay systems using techniques
such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope
labels, for example), western blots, precipitation reactions, agglutination
assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation assays,
immunofluorescence assays, protein A assays, and immunoelectrophoresis assays,
etc. In one
embodiment, antibody binding is detected by detecting a label on the primary
antibody. In
another embodiment, the primary antibody is detected by detecting binding of a
secondary
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antibody or reagent to the primary antibody. In a further embodiment, the
secondary
antibody is labeled. Many means are known in the art for detecting binding in
an
immunoassay and are within the scope of the present invention.
[111 ] In another embodiment, where a ligand is identified, or the ability of
a polypeptide
fragment, variant or derivative of the invention to multimerize is being
evaluated, binding
can be assayed, e.g., by means well-known in the art, such as, for example,
reducing and non-
reducing gel chromatography, protein affinity chromatography, and affinity
blotting. See
generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another
embodiment, the
ability of physiological correlates of a polypeptide of the present invention
to bind to a
substrates) of the polypeptide of the invention can be routinely assayed using
techniques
known in the art.
[112] In addition, assays described herein (see Examples) and otherwise known
in the art
may routinely be applied to measure the ability of polypeptides of the present
invention and
fragments, variants and derivatives thereof to elicit polypeptide related
biological activity
(either in vitro or in vivo). Other methods will be known to the skilled
artisan and are within
the scope of the invention.
(113] Of course, due to the degeneracy of the genetic code, one of ordinary
skill in the
art will immediately recognize that a large number of the nucleic acid
molecules having a
sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to, for
example, the nucleic acid sequence of the cDNA contained in Clone ID NO:Z, the
nucleic
acid sequence referred to in Table 1A (SEQ ID NO:X), the nucleic acid sequence
disclosed
in Table 2 (e.g,. the nucleic acid sequence delineated in columns 8 and 9) or
fragments
thereof, will encode polypeptides "having functional activity." In fact, since
degenerate
variants of any of these nucleotide sequences all encode the same polypeptide,
in many
instances, this will be clear to the skilled artisan even without performing
the above described
comparison assay. It will be further recognized in the art that, for such
nucleic acid
molecules that are not degenerate variants, a reasonable number will also
encode a
polypeptide having functional activity. This is because the skilled artisan is
fully aware of
amino acid substitutions that are either less likely or not likely to
significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second aliphatic
amino acid), as
further described below.
[114] For example, guidance concerning how to make phenotypically silent amino
acid
substitutions is provided in Bowie et al., "Deciphering the Message in Protein
Sequences:
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Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein
the authors
indicate that there are two main strategies for studying the tolerance of an
amino acid
sequence to change.
[115] The first strategy exploits the tolerance of amino acid substitutions by
natural
selection during the process of evolution. By comparing amino acid sequences
in different
species, conserved amino acids can be identified. These conserved amino acids
are likely
important for protein function. In contrast, the amino acid positions where
substitutions have
been tolerated by natural selection indicates that these positions are not
critical for protein
function. Thus, positions tolerating amino acid substitution could be modified
while still
maintaining biological activity of the protein.
[116J The second strategy uses genetic engineering to introduce amino acid
changes at
specific positions of a cloned gene to identify regions critical for protein
function. For
example, site directed mutagenesis or alanine-scanning mutagenesis
(introduction of single
alanine mutations at every residue in the molecule) can be used. See
Cunningham and Wells,
Science 244:1081-1085 (1989). The resulting mutant molecules can then be
tested for
biological activity.
[117] As the authors state, these two strategies have revealed that proteins
are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which amino
acid changes are likely to be permissive at certain amino acid positions in
the protein. For
example, most buried (within the tertiary structure of the protein) amino acid
residues require
nonpolar side chains, whereas few features of surface side chains are
generally conserved.
Moreover, tolerated conservative amino acid substitutions involve replacement
of the
aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the
hydroxyl
residues Ser and Thr; replacement of the acidic residues Asp and Glu;
replacement of the
amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and
His;
replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the
small-sized
amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid
substitution,
variants of the present invention include (i) substitutions with one or more
of the non-
conserved amino acid residues, where the substituted amino acid residues may
or may not be
one encoded by the genetic code, or (ii) substitutions with one or more of the
amino acid
residues having a substituent group, or (iii) fusion of the mature polypeptide
with another
compound, such as a compound to increase the stability and/or solubility of
the polypeptide
(for example, polyethylene glycol), (iv) fusion of the polypeptide with
additional amino
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acids, such as, for example, an IgG Fc fusion region peptide, serum albumin
(preferably
human serum albumin) or a fragment thereof, or leader or secretory sequence,
or a sequence
facilitating purification, or (v) fusion of the polypeptide with another
compound, such as
albumin (including but not limited to recombinant albumin (see, e.g., U.S.
Patent No.
5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
5,766,883, issued
June 16, 1998, herein incorporated by reference in their entirety)). Such
variant
polypeptides are deemed to be within the scope of those.skilled in the art
from the teachings
herein.
[118] For example, polypeptide variants containing amino acid substitutions of
charged
amino acids with other charged or neutral amino acids may produce proteins
with improved
characteristics, such as less aggregation. Aggregation of pharmaceutical
formulations both
reduces activity and increases clearance due to the aggregate's immunogenic
activity. See
Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al.,
Diabetes 36: 838-845
(1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377
(1993).
[119] A further embodiment of the invention relates to polypeptides which
comprise the
amino acid sequence of a polypeptide having an amino acid sequence which
contains at least
one amino acid substitution, but not more than 50 amino acid substitutions,
even more
preferably, not more than 40 amino acid substitutions, still more preferably,
not more than 30
amino acid substitutions, and still even more preferably, not more than 20
amino acid
substitutions from a polypeptide sequence disclosed herein. Of course it is
highly preferable
for a polypeptide to have an amino acid sequence which comprises the amino
acid sequence
of a polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID
NO:X, an
amino acid sequence encoded by the portion of SEQ 1D NO:X as defined in
columnns 8 and
9 of Table 2, an amino acid sequence encoded by the complement of SEQ ID NO:X,
and/or
an amino acid sequence encoded by eDNA contained in Clone >D NO:Z which
contains, in
order of ever-increasing preference, at least one, but not more than 10, 9, 8,
7, 6, 5, 4, 3, 2 or
1 amino acid substitutions.
[120] In specific embodiments, the polypeptides of the invention comprise, or
alternatively, consist of, fragments or variants of a reference amino acid
sequence selected
from: (a) the amino acid sequence of SEQ )D NO:Y or fragments thereof (e.g.,
the mature
form and/or other fragments described herein); (b) the amino acid sequence
encoded by SEQ
>D NO:X or fragments thereof; (c) the amino acid sequence encoded by the
complement of
SEQ )T7 NO:X or fragments thereof; (d) the amino acid sequence encoded by the
portion of
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SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and
(e) the
amino acid sequence encoded by cDNA contained in Clone >D NO:Z or fragments
thereof;
wherein the fragments or variants have 1-S, S-10, S-2S, S-S0, 10-SO or SO-150,
amino acid
residue additions, substitutions, and/or deletions when compared to the
reference amino acid
sequence. In preferred embodiments, the amino acid substitutions are
conservative.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Polynucleotide and Polvpeptide Fragments
[121] The present invention is also directed to polynucleotide fragments of
the
polynucleotides (nucleic acids) of the invention. In the present invention, a
"polynucleotide
fragment" refers to a polynucleotide having a nucleic acid sequence which, for
example: is a
portion of the cDNA contained in Clone >D NO:Z or the complementary strand
thereto; is a
portion of the polynucleotide sequence encoding the polypeptide encoded by the
cDNA
contained in Clone ID NO:Z or the complementary strand thereto; is a portion
of a
polynucleotide sequence encoding the amino acid sequence encoded by the region
of SEQ >D
NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand
thereto; is a
portion of the polynucleotide sequence of SEQ >D NO:X as defined in columns 8
and 9 of
Table 2 or the complementary strand thereto; is a portion of the
polynucleotide sequence in
SEQ >D NO:X or the complementary strand thereto; is a polynucleotide sequence
encoding a
portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence
encoding a portion
of a polypeptide encoded by SEQ >D NO:X; is a polynucleotide sequence encoding
a portion
of a polypeptide encoded by the complement of the polynucleotide sequence in
SEQ >D
NO:X; is a portion of a polynucleotide sequence encoding the amino acid
sequence encoded
by the region of SEQ 1D NO:B as defined in column 6 of Table 1B or the
complementary
strand thereto; or is a portion of the polynucleotide sequence of SEQ m NO:B
as defined in
column 6 of Table 1 B or the complementary strand thereto.
[122] The polynucleotide fragments of the invention are preferably at least
about 1S nt,
and more preferably at least about 20 nt, still more preferably at least about
30 nt, and even
more preferably, at least about 40 nt, at least about 50 nt, at least about 7S
nt, or at least about
1 SO nt in length. A fragment "at least 20 nt in length," for example, is
intended to include 20
or more contiguous bases from the cDNA sequence contained in Clone >D NO:Z, or
the
nucleotide sequence shown in SEQ 117 NO:X or the complementary stand thereto.
In this
context "about" includes the particularly recited value or a value larger or
smaller by several
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(5, 4, 3, 2, or 1 ) nucleotides, at either terminus or at both termini. These
nucleotide
flragments have uses that include, but are not limited to, as diagnostic
probes and primers as
discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180,
190, 200, 250,
500, 600, 1000, or 2000 nucleotides in length ) are also encompassed by the
invention.
[123] Moreover, representative examples of polynucleotide fragments of the
invention
comprise, or alternatively consist of, a sequence from about nucleotide number
1-50, 51-100,
101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-
550, 551-
600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000,
1001-
1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-
1400,
1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750,
1751-
1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-
2150,
2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500,
2501-
2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-
2900,
2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250,
3251-
3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-
3650,
3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000,
4001-
4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-
4400,
4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750,
4751-
4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-
5150,
5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500,
5501-
5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-
5900,
5901-5950, 5951-6000, 6001-6050, ,6051-6100, 6101-6150, 6151-6200, 6201-6250,
6251-
6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-
6650,
6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000,
7001-
7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end
of SEQ
>D NO:X, or the complementary strand thereto. In this context "about" includes
the
particularly recited range or a range larger or smaller by several (5, 4, 3,
2, or 1) nucleotides,
at either terminus or at both termini. Preferably, these fragments encode a
polypeptide which
has a functional activity (e.g., biological activity). More preferably, these
polynucleotides
can be used as probes or primers as discussed herein. Polynucleotides which
hybridize to one
or more of these polynucleotides under stringent hybridization conditions or
alternatively,
under lower stringency conditions are also encompassed by the invention, as
are polypeptides
encoded by these polynucleotides.
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[124] Further representative examples of polynucleotide fragments of the
invention
Comprise, or alternatively consist of, a sequence from about nucleotide number
1-50, 51-100,
101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-
550, 551-
600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000,
1001-
1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-
1400,
1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750,
1751-
1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-
2150,
2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500,
2501-
2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-
2900,
2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250,
3251-
3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-
3650,
3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000,
4001-
4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-
4400,
4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750,
4751-
4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-
515.0,
5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500,
5501-
5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-
5900,
5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250,
6251-
6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-
6650,
6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000,
7001-
7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end
of the
cDNA sequence contained in Clone >D NO:Z, or the complementary strand thereto.
In this
context "about" includes the particularly recited range or a range larger or
smaller by several
(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferably, these fragments
encode a polypeptide which has a functional activity (e.g., biological
activity). More
preferably, these polynucleotides can be used as probes or primers as
discussed herein.
Polynucleotides which hybridize to one or more of these polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions
are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
[125] Moreover, representative examples of polynucleotide fragments of the
invention
comprise, or alternatively consist of, a nucleic acid sequence comprising one,
two, three,
four, five, six, seven, eight, nine, ten, or more of the above described
polynucleotide
fragments of the invention in combination with a polynucleotide sequence
delineated in
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Table 1B column 6. Additional, representative examples of polynucleotide
fragments of the
invention comprise, or alternatively consist of, a nucleic acid sequence
comprising one, two,
three, four, five, six, seven, eight, nine, ten, or more of the above
described polynucleotide
fragments of the invention in combination with a polynucleotide sequence that
is the
complementary strand of a sequence delineated in column 6 of Table 1B. In
further
embodiments, the above-described polynucleotide fragments of the invention
comprise, or
alternatively consist of, sequences delineated in Table 1B, column 6, and have
a nucleic acid
sequence which is different from that of the BAC fragment having the sequence
disclosed in
SEQ >D NO:B (see Table 1 B, column 5). In additional embodiments, the above-
described
polynucleotide fragments of the invention comprise, or alternatively consist
of, sequences
delineated in Table 1B, column 6, and have a nucleic acid sequence which is
different from
that published for the BAC clone identified as BAC >D NO:A (see Table 1B,
column 4). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist of; sequences delineated Table 1B, column 6, and have a
nucleic acid
sequence which is different from that contained in the BAC clone identified as
BAC ID
NO:A (see Table 1 B, column 4). Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
are also encompassed by the invention. Additionally, fragments and variants of
the above-
described polynucleotides and polypeptides are also encompassed by the
invention.
[126] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in column 6 of Table 1B, and the
polynucleotide
sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column 2) or fragments
or variants
thereof. Polypeptides encoded by these polynucleotides, other polynucleotides
that encode
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention.
[127J In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in column 6 of Table 1B which correspond
to the same
Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ
1!D NO:X
(e.g., as defined in Table 1A or 1B) or fragments or variants thereof.
Polypeptides encoded
by these polynucleotides, other polynucleotides that encode these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
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[128] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in the same row of column 6 of Table 1B,
and the
polynucleotide sequence of SEQ >D NO:X (e.g., as defined in Table 1A or 1B) or
fragments
or variants thereof. Polypeptides encoded by these polynucleotides, other
polynucleotides
that encode these polypeptides, and antibodies that bind these polypeptides
are also
encompassed by the invention.
[129] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of the
sequence of~ SEQ ID NO:X are directly contiguous. Nucleic acids which
hybridize to the
complement of these 20 contiguous polynucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids that encode these polypeptides, and antibodies that bind
these
polypeptides are also encompassed by the invention. Additionally, fragments
and variants of
the above-described polynucleotides, nucleic acids, and polypeptides are also
encompassed
by the invention.
[130) In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of a
fragment or variant of the sequence of SEQ >D NO:X (e.g., as described herein)
are directly
contiguous Nucleic acids which hybridize to the complement of these 20
contiguous
polynucleotides under stringent hybridization conditions or alternatively,
under lower
stringency conditions, are also encompassed by the invention. Polypeptides
encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids encoding
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention. Additionally, fragments and variants of the above-described
polynucleotides,
nucleic acids, and polypeptides are also encompassed by the invention.
[131] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of a
fragment or variant of the sequence of SEQ >l7 NO:X and the S' 10
polynucleotides of the
sequence of one of the sequences delineated in column 6 of Table 1B are
directly contiguous.
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Nucleic acids which hybridize to the complement of these 20 contiguous
polynucleotides
under stringent hybridization conditions or alternatively, under lower
stringency conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides
and/or nucleic acids, other polynucleotides and/or nucleic acids encoding
these polypeptides,
and antibodies that bind these polypeptides are also encompassed by the
invention.
Additionally, fragments and variants of the above-described polynucleotides,
nucleic acids,
and polypeptides are also encompassed by the invention.
[132] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another
sequence in column 6 are directly contiguous. In preferred embodiments, the 3'
10
polynucleotides of one of the sequences delineated in column 6 of Table 1B is
directly
contiguous with the 5' 10 polynucleotides of the next sequential exon
delineated in Table 1B,
column 6. Nucleic acids which hybridize to the complement of these 20
contiguous
polynucleotides under stringent hybridization conditions or alternatively,
under lower
stringency conditions, are also encompassed by the invention. Polypeptides
encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids encoding
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention. Additionally, fragments and variants of the above-described
polynucleotides,
nucleic acids, and polypeptides are also encompassed by the invention.
[133] In the present invention, a "polypeptide fragment" refers to an amino
.acid
sequence which is a portion of that contained in SEQ >D NO:Y, a portion of an
amino acid
sequence encoded by the portion of SEQ >D NO:X as defined in columnns 8 and 9
of Table
2, a portion of an amino acid sequence encoded by the polynucleotide sequence
of SEQ m
NO:X, a portion of an amino acid sequence encoded by the complement of the
polynucleotide sequence in SEQ m NO:X, and/or a portion of an amino acid
sequence
encoded by the cDNA contained in Clone >D NO:Z. Protein (polypeptide)
fragments may be
"free-standing," or comprised within a larger polypeptide of which the
fragment forms a part
or region, most preferably as a single continuous region. Representative
examples of
polypeptide fragments of the invention, include, for example, fragments
comprising, or
alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-100,
101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-
280, 281-
300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460,
461-480,
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481-500, 501-520, 521-540. 541-560, 561-580, 581-600, 601-620, 621-640, 641-
660, 661-
680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840,
841-860,
861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020,
1021-1040,
1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180,
1181-
1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-
1340,
1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of
the coding
region of cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptide
fragments of
the invention include, for example, fragments comprising, or alternatively
consisting of, from
about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140,
141-160,
161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-
340, 341-
360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520,
521-540,
541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-
720, 721-
740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900,
901-920,
921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-
1080, 1081-
1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-
1240,
1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380,
1381-
1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID
NO:Y.
Moreover, polypeptide fragments of the invention may be at least about 10, 15,
20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or
150 amino acids in
length. In this context "about" includes the particularly recited ranges or
values, or ranges or
values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either
extreme or at both
extremes. Polynucleotides encoding these polypeptide fragments are also
encompassed by
the invention.
[134] Even if deletion of one or more amino acids from the N-terminus of a
protein
results in modification of loss of one or more biological functions of the
protein, other
functional activities (e.g., biological activities, ability to multimerize,
ability to bind a ligand)
may still be retained. For example, the ability of shortened muteins to induce
and/or bind to
antibodies which recognize the complete or mature forms of the polypeptides
generally will
be retained when less than the majority of the residues of the complete or
mature polypeptide
are removed from the N-terminus. Whether a particular polypeptide lacking N-
terminal
residues of a complete polypeptide retains such immunologic activities can
readily be
determined by routine methods described herein and otherwise known in the art.
It is not
unlikely that a mutein with a large number of deleted N-terminal amino acid
residues may
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retain some biological or immunogenic activities. In fact, peptides composed
of as few as six
amino acid residues may often evoke an immune response.
[135] Accordingly, polypeptide fragments include the secreted protein as well
as the
mature form. Further preferred polypeptide fragments include the secreted
protein or the
mature form having a continuous series of deleted residues from the amino or
the carboxy
terminus, or both. For example, any number of amino acids, ranging from 1-60,
can be
deleted from the amino terminus of either the secreted polypeptide or the
mature form.
Similarly, any number of amino acids, ranging from 1-30, can be deleted from
the carboxy
terminus of the secreted protein or mature form. Furthermore, any combination
of the above
amino and carboxy terminus deletions are preferred. Similarly, polynucleotides
encoding
these polypeptide fragments are also preferred.
[136] The present invention further provides polypeptides having one or more
residues
deleted from the amino terminus of the amino acid sequence of a polypeptide
disclosed
herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the
polynucleotide
sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide
encoded by
the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a
polypeptide
encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1B,
and/or a
polypeptide encoded by the cDNA contained in Clone ID NO:Z). In particular, N-
terminal
deletions may be described by the general formula m-q, where q is a whole
integer
representing the total number of amino acid residues in a polypeptide of the
invention (e.g.,
the polypeptide disclosed in SEQ ID NO:Y, or the polypeptide encoded by the
portion of
SEQ 117 NO:X as defined in columns 8 and 9 of Table 2), and m is defined as
any integer
ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also
encompassed by
the invention.
[137] The present invention further provides polypeptides having one or more
residues
from the carboxy terminus of the amino acid sequence of a polypeptide
disclosed herein (e.g.,
a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide
sequence
contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ ID NO:X
as
defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by the
cDNA contained
in Clone >D NO:Z). In particular, C-terminal deletions may be described by the
general
formula I-n, where n is any whole integer ranging from 6 to q-I, and where n
corresponds to
the position of amino acid residue in a polypeptide of -the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
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[138] In addition, any of the above described N- or C-terminal deletions can
be
trombined to produce a N- and C-terminal deleted polypeptide. The invention
also provides
polypeptides having one or more amino acids deleted from both the amino and
the carboxyl
termini, which may be described generally as having residues m-n of a
polypeptide encoded
by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide
disclosed as
SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ >Z7 NO:X as
defined in
columns 8 and 9 of Table 2), the cDNA contained in Clone >D NO:Z, and/or the
complement
thereof, where n and m are integers as described above. Polynucleotides
encoding these
polypeptides are also encompassed by the invention.
[139] ~ Also as mentioned above, even if deletion of one or more amino acids
from the
C-terminus of a protein results in modification of loss of one or more
biological functions of
the protein, other functional activities (e.g., biological activities, ability
to multimerize,
ability to bind a ligand) may still be retained. For example the ability of
the shortened mutein
to induce and/or bind to antibodies which recognize the complete or mature
forms of the
polypeptide generally will be retained when less than the majority of the
residues of the
complete or mature polypeptide are removed from the C-terminus. Whether a
particular
polypeptide lacking C-terminal residues of a complete polypeptide retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise
known in the art. It is not unlikely that a mutein with a large number of
deleted C-terminal
amino acid residues may retain some biological or immunogenic activities. In
fact, peptides .
composed of as few as six amino acid residues may often evoke an immune
response.
[140] The present application is also directed to proteins containing
polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence
set forth
herein. In preferred embodiments, the application is directed to proteins
containing
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
polypeptides
having the amino acid sequence of the specific N- and C-terminal deletions.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[141], Any polypeptide sequence encoded by, for example, the polynucleotide
sequences
set forth as SEQ 1D NO:X or the complement thereof, (presented, for example,
in Tables 1A
and 2), the cDNA contained in Clone ID NO:Z, or the polynucleotide sequence as
defined in
column 6 of Table 1 B, may be analyzed to determine certain preferred regions
of the
polypeptide. For example, the amino acid sequence of a polypeptide encoded by
a
polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ >D NO:Y
and the
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polypeptide encoded by the portion of SEQ ID NO:X as defined in columnns 8 and
9 of
Fable 2) or the cDNA contained in Clone ID NO:Z may be analyzed using the
default
parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St.,
Madison, WI 53715 USA; http://www.dnastar.com/).
[142] Polypeptide regions that may be routinely obtained using the DNASTAR
computer
algorithm include, but are not limited to, Gamier-Robson alpha-regions, beta-
regions,
turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and
turn-regions;
Kyte-Doolittle hydrophilic regions and hydrophobic regions; Eisenberg alpha-
and
beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-
forming regions;
and Jameson-Wolf regions of high antigenic index. Among highly preferred
polynucleotides
of the invention in this regard are those that encode polypeptides comprising
regions that
combine several structural features, such as several (e.g., l, 2, 3 or 4) of
the features set out
above.
[143] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic
regions, Emini
surface-forming regions, and Jameson-Wolf regions of high antigenic index
(i.e., containing
four or more contiguous amino acids having an antigenic index of greater than
or equal to
1.5, as identified using the default parameters of the Jameson-Wolf program)
can routinely be
used to determine polypeptide regions that exhibit a high degree of potential
for antigenicity.
Regions of high antigenicity are determined from data by DNASTAR analysis by
choosing
values which represent regions of the polypeptide which are likely to be
exposed on the
surface of the polypeptide in an environment in which antigen recognition may
occur in the
process of initiation of an immune response.
[144] Preferred polypeptide fragments of the invention are fragments
comprising, or
alternatively, consisting of, an amino acid sequence that displays a
functional activity (e.g.
biological activity) of the polypeptide sequence of which the amino acid
sequence is a
fragment. By a polypeptide displaying a "functional activity" is meant a
polypeptide capable
of one or more known functional activities associated with a full-length
protein, such as, for
example, biological activity, antigenicity, immunogenicity, and/or
multimerization, as
described herein.
[145] Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not
necessarily
identical,.to an activity of the polypeptide of the present invention. The
biological activity of
the fragments may include an improved desired activity, or a decreased
undesirable activity.
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[146] In preferred embodiments, polypeptides of the invention comprise, or
alternatively
consist of, one, two, three, four, five or more of the antigenic fragments of
the polypeptide of
SEQ )D NO:Y, or portions thereof. Polynucleotides encoding these polypeptides
are also
encompassed by the invention.
[147] The present invention encompasses polypeptides comprising, or
alternatively
consisting of, an epitope of: the polypeptide sequence shown in SEQ m NO:Y; a
polypeptide
sequence encoded by SEQ ID NO:X or the complementary strand thereto; the
polypeptide
sequence encoded by the portion of SEQ >D NO:X as defined in columns 8 and 9
of Table 2;
the polypeptide sequence encoded by the portion of SEQ m NO:B as defined in
column 6 of
Table 1B or the complement thereto; the polypeptide sequence encoded by the
eDNA
contained in Clone ID NO:Z; or the polypeptide sequence encoded by a
polynucleotide that
hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of
SEQ 1D
NO:X, the complement of a portion of SEQ >D NO:X as defined in columns 8 and 9
of Table
2, or the cDNA sequence contained in Clone 1D NO:Z under stringent
.hybridization
conditions or alternatively, under lower stringency hybridization as defined
supra. The
present invention further encompasses polynucleotide sequences encoding an
epitope of a
polypeptide sequence o f the invention (such as, for example, the sequence
disclosed in SEQ
)D NO:X, or a fragment thereof), polynucleotide sequences of the complementary
strand of a
polynucleotide sequence encoding an epitope of the invention, and
polynucleotide sequences
which hybridize to the complementary strand under stringent hybridization
conditions or
alternatively, under lower stringency hybridization conditions defined supra.
[148] The term "epitopes," as used herein, refers to portions of a polypeptide
having
antigenic or immunogenic activity in an animal, preferably a mammal, and most
preferably
in a human. In a preferred embodiment, the present invention encompasses a
polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An
"immunogenic epitope," as used herein, is defined as a portion of a protein
that elicits an
antibody response in an animal, as determined by any method known in the art,
for example,
by the methods for generating antibodies described infra. (See, for example,
Geysen et al.,
Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983)). The term "antigenic
epitope," as used
herein, is defined as a portion of a protein to which an antibody can
immunospecifically bind
its antigen as determined by any method well known in the art, for example, by
the
immunoassays described herein. Immunospecific binding excludes non-specific
binding but
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does not necessarily exclude cross- reactivity with other antigens. Antigenic
epitopes need
hot necessarily be immunogenic.
(149] Fragments which function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985) further
described in U.S. Patent No. 4,631,211.)
[150] In the present invention, antigenic epitopes preferably contain a
sequence of at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 1 S, at least 20, at least
25, at least 30, at least
40, at least 50, and, most preferably, between about 15 to about 30 amino
acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15,
20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues
in length.
Additional non-exclusive preferred antigenic epitopes include the antigenic
epitopes
disclosed herein, as well as portions thereof. Antigenic epitopes are useful,
for example, to
raise antibodies, including monoclonal antibodies, that specifically bind the
epitope.
Preferred antigenic epitopes include the antigenic epitopes disclosed herein,
as well as any
combination of two, three, four, five or more of these antigenic epitopes.
Antigenic epitopes
can be used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell
37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
[151] Non-limiting examples of epitopes of polypeptides that can be used to
generate
antibodies of the invention include a polypeptide comprising, or alternatively
consisting of,
at least one, two, three, four, five, six or more of the portions) of SEQ m
NO:Y specified in
column 7 of Table 1 A. These polypeptide fragments have been determined to
bear antigenic
epitopes of the proteins of the invention by the analysis of the Jameson-Wolf
antigenic index
which is included in the DNAStar suite of computer programs. By "comprise" it
is intended
that a polypeptide contains at least one, two, three, four, five, six or more
of the portions) of
SEQ >D NO:Y shown in column 7 of Table 1A, but it may contain additional
flanking
residues on either the amino or carboxyl termini of the recited portion. Such
additional
flanking sequences are preferably sequences naturally found adjacent to the
portion; i.e.,
contiguous sequence shown in SEQ >D NO:Y. The flanking sequence may, however,
be
sequences from a heterolgous polypeptide, such as from another protein
described herein or
from a heterologous polypeptide not described herein. In particular
embodiments, epitope
portions of a polypeptide of the invention comprise one, two, three, or more
of the portions of
SEQ >D NO:Y shown in column 7 of Table 1A.
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[152] Similarly, immunogenic epitopes can be used, for example, to induce
antibodies
according to methods well known in the art. See, for instance, Sutcliffe et
al., supra; Wilson
et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle
et al., J. Gen.
Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the
immunogenic
epitopes disclosed herein, as well as any combination of two, three, four,
five or more of
these immunogenic epitopes. The polypeptides comprising one or more
immunogenic
epitopes may be presented for eliciting an antibody response together with a
carrier protein,
such as an albumin, to an animal system (such as rabbit or mouse), or, if the
polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide may be
presented without a
carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino
acids have
been shown to be sufficient to raise antibodies capable of binding to, at the
very least, linear
epitopes in a denatured polypeptide (e.g., in Western blotting).
[153] Epitope-bearing polypeptides of the present invention may be used to
induce
antibodies according to methods well known in the art including, but not
limited to, in vivo
immunization, in vitro immunization, and phage display methods. See, e.g.,
Sutcliffe et al.,
supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354
(1985). If in vivo
immunization is used, animals may be immunized with free peptide; however,
anti-peptide
antibody titer may be boosted by coupling the peptide to a macromolecular
earner, such as
keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides
containing
cysteine residues may be coupled to a carrier using a linker such as
maleimidobenzoyl- N-
hydroxysuccinimide ester (MBS), while other peptides may be coupled to earners
using a
more general linking agent such as glutaraldehyde. Animals such as rabbits,
rats and mice
are immunized with either free or earner- coupled peptides, for instance, by
intraperitoneal
and/or intradermal injection of emulsions containing about 100 pg of peptide
or carrier
protein and Freund's adjuvant or any other adjuvant known for stimulating an
immune
response. Several booster injections may be needed, for instance, at intervals
of about two
weeks, to provide a useful titer of anti-peptide antibody which can be
detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The titer of
anti-peptide
antibodies in serum from an immunized animal may be increased by selection of
anti-peptide
antibodies, for instance, by adsorption to the peptide on a solid support and
elution of the
selected antibodies according to methods well known in the art.
[154] As one of skill in the art will appreciate, and as discussed above, the
polypeptides
of the present invention (e.g., those comprising an immunogenic or antigenic
epitope) can be
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fused to heterologous polypeptide sequences. For example, polypeptides of the
present
invention (including fragments or variants thereof), may be fused with the
constant domain of
immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or
any
combination thereof and portions thereof, resulting in chimeric polypeptides.
By way of
another non-limiting example, polypeptides and/or antibodies of the present
invention
(including fragments or variants thereof) may be fused with albumin (including
but not
limited to recombinant human serum albumin or fragments or variants thereof
(see, e.g., U.S.
Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S.
Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their
entirety)). In a
preferred embodiment, polypeptides and/or antibodies of the present invention
(including
fragments or variants thereof) are fused with the mature form of human serum
albumin (i.e.,
amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0 322
094) which is herein incorporated by reference in its entirety. In another
preferred
embodiment, polypeptides and/or antibodies of the present invention (including
fragments or
variants thereof) are fused with polypeptide fragments comprising, or
alternatively consisting
of, amino acid residues 1-z of human serum albumin, where z is an integer from
369 to 419,
as described in U.S. Patent 5,766,883 herein incorporated by reference in .its
entirety.
Polypeptides and/or antibodies of the present invention (including fragments
or variants
thereof) may be fused to either the N- or C-terminal end of the heterologous
protein (e.g.,
immunoglobulin Fc polypeptide or human serum albumin polypeptide).
Polynucleotides
encoding fusion proteins of the invention are also encompassed by the
invention.
[155] Such fusion proteins as those described above may facilitate
purification and may
increase half life in vivo. This has been shown for chimeric proteins
consisting of the first
two domains of the human CD4-polypeptide and various domains of the constant
regions of
the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen
across the
epithelial barrier to the immune system has been demonstrated for antigens
(e.g., insulin)
conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g.,
PCT
Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a
disulfide-
linked dimeric structure due to the IgG portion desulfide bonds have also been
found to be
more efficient in binding and neutralizing other molecules than monomeric
polypeptides or
fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-
3964 (1995).
Nucleic acids encoding the above epitopes can also be recombined with a gene
of interest as
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an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in
detection and
purification of the expressed polypeptide. For example, a system described by
Janknecht et
al. allows for the ready purification of non-denatured fusion proteins
expressed in human
cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897).
In this system,
the gene of interest is subcloned into a vaccinia recombination plasmid such
that the open
reading frame of the gene is translationally fused to an amino-terminal tag
consisting of six
histidine residues. The tag serves as a matrix binding domain for the fusion
protein.
Extracts from cells infected with the recombinant vaccinia virus are loaded
onto Ni2+
nitriloacetic acid-agarose column and histidine-tagged proteins can be
selectively eluted with
imidazole-containing buffers.
Fusion Proteins
[156] Any polypeptide of the present invention can be used to generate fusion
proteins.
For example, the polypeptide of the present invention, when fused to a second
protein, can be
used as an antigenic tag. Antibodies raised against the polypeptide of the
present invention
can be used to indirectly detect the second protein by binding to the
polypeptide. Moreover,
because secreted proteins target cellular locations based on trafficking
signals, polypeptides
of the present invention which are shown to be secreted can be used as
targeting molecules
once fused to other proteins.
(157] Examples of domains that can be fused to polypeptides of the present
invention
include not only heterologous signal sequences, but also other heterologous
functional
regions. The fusion does not necessarily need to be direct, but may occur
through linker
sequences.
[158] In certain preferred embodiments, proteins of the invention are fusion
proteins
comprising an amino acid sequence that is an N and/or C- terminal deletion of
a polypeptide
of the invention. In preferred embodiments, the invention is directed to a
fusion protein
comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or
99%
identical to a polypeptide sequence of the invention. Polynucleotides encoding
these proteins
are also encompassed by the invention.
[159] Moreover, fusion proteins may also be engineered to improve
characteristics of the
polypeptide of the present invention. For instance, a region of additional
amino acids,
particularly charged amino acids, may be added to the N-terminus of the
polypeptide to
improve stability and persistence during purification from the host cell or
subsequent
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handling and storage. Also, peptide moieties may be added to the polypeptide
to facilitate
purification. Such regions may be removed prior to final preparation of the
polypeptide. The
addition of peptide moieties to facilitate handling of polypeptides are
familiar and routine
techniques in the art.
[160] As one of skill in the art will appreciate that, as discussed above,
polypeptides of
the present invention, and epitope-bearing fragments thereof, can be combined
with
heterologous polypeptide sequences. For example, the polypeptides of the
present invention
may be fused with heterologous polypeptide sequences, for example, the
polypeptides of the
present invention may be fused with the constant domain of immunoglobulins
(IgA, IgE,
IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof,
including both
entire domains and portions thereof), or albumin (including, but not limited
to, native or
recombinant human albumin or fragments or variants thereof (see, e.g., U.S.
Patent No.
5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
5,766,883, issued
June 16, 1998, herein incorporated by reference in their entirety)), resulting
in chimeric
polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses
fusion proteins comprising various portions of constant region of
immunoglobulin molecules
together with another human protein or part thereof. In many cases, the Fc
part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result in, for
example, improved
pharmacokinetic properties (EP-A 0232 262). Alternatively, deleting the Fc
part after the
fusion protein has been expressed, detected, and purified, would be desired.
For example, the
Fc portion may hinder therapy and diagnosis if the fusion protein is used as
an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have been
fused with Fc portions for the purpose of high-throughput screening assays to
identify
antagonists of hIL-5. See, D. Bennett et al., J. Molecular Recognition 8:52-58
(1995); K.
Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[161] Moreover, the polypeptides of the present invention can be fused to
marker
sequences, such as a polypeptide which facilitates purification of the fused
polypeptide. In
preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
CA,
91311), among others, many of which are commercially available. As described
in Gentz et
al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for
convenient purification of the fusion protein. Another peptide tag useful for
purification, the
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"HA" tag, corresponds to an epitope derived from the influenza hemagglutinin
protein
Wilson et al., Cell 37:767 (1984)).
[162] Additional fusion proteins of the invention may be generated through the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to modulate
the activities of polypeptides of the invention, such methods can be used to
generate
polypeptides with altered activity, as well as agonists and antagonists of the
polypeptides.
See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252;
and 5,837,458,
and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends
Biotechnol.
16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and
Lorenzo and
Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and
publications are
hereby incorporated by reference in its entirety). In one embodiment,
alteration of
polynucleotides corresponding to SEQ >D NO:X and the polypeptides encoded by
these
polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the
assembly
of two or more DNA segments by homologous or site-specific recombination to
generate
variation in the polynucleotide sequence. In another embodiment,
polynucleotides of the
invention, or the encoded polypeptides, may be altered by being subjected to
random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior to
recombination. In another embodiment, one or more components, motifs,
sections, parts,
domains, fragments, etc., of a polynucleotide encoding a polypeptide of the
invention may be
recombined with one or more components, motifs, sections, parts, domains,
fragments, etc.
of one or more heterologous molecules.
[163] Thus, any of these above fusions can be engineered using the
polynucleotides or
the polypeptides of the present invention.
Recombinant and Synthetic Production of Pol'rpe~tides of the Invention
[164] The present invention also relates to vectors containing the
polynucleotide of the
present invention, host cells, and the production of polypeptides by synthetic
and
recombinant techniques. The vector may be, for example, a phage, plasmid,
viral, or
retroviral vector. Retroviral vectors may be replication competent or
replication defective.
In the latter case, viral propagation generally will occur only in
complementing host cells.
[165] The polynucleotides of the invention may be joined to a vector
containing a
selectable marker for propagation in a host. Generally, a plasmid vector is
introduced in a
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precipitate, such as a calcium phosphate precipitate, or in a complex with a
charged lipid. If
the vector is a virus, it may be packaged in vitro using an appropriate
packaging cell line and
then transduced into host cells.
[166] The polynucleotide insert should be operatively linked to an appropriate
promoter,
such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac
promoters, the SV40
early and late promoters and promoters of retroviral LTRs, to name a few.
Other suitable
promoters will be known to the skilled artisan. The expression constructs will
further contain
sites for transcription initiation, termination, and, in the transcribed
region, a ribosome
binding site for translation. The coding portion of the transcripts expressed
by the constructs
will preferably include a translation initiating codon at the beginning and a
termination codon
(UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be
translated.
[167] As indicated, the expression vectors will preferably include at least
one selectable
marker. Such markers include dihydrofolate reductase, 6418, glutamine
synthase, or
neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin
or ampicillin
resistance genes for culturing in E. coli and other bacteria. Representative
examples of
appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces
and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g.,
Saccharomyces
cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such
as Drosophila
S2 and Spodoptera Sf~ cells; animal cells such as CHO, COS, 293, and Bowes
melanoma
cells; and plant cells. Appropriate culture mediums and conditions for the
above-described
host cells are known in the art.
[168] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-
9,
available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA,
pNHl6a,
pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK223-
3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among
preferred
eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from
Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred
expression vectors for use in yeast systems include, but are not limited to
pYES2, pYDI,
pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-
S1,
pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA).
Other
suitable vectors will be readily apparent to the skilled artisan.
[169] Vectors which use glutamine synthase (GS) or DHFR as the selectable
markers
can be amplified in the presence of the drugs methionine sulphoximine or
methotrexate,
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respectively. An advantage of glutamine synthase based vectors are the
availabilty of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase
negative.
Glutamine synthase expression systems can also function in glutamine synthase
expressing
cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional
inhibitor to prevent
the functioning of the endogenous gene. A glutamine synthase expression system
and
components thereof are detailed in PCT publications: W087/04462; W086/05807;
W089/01036; W089/10404; and W091/06657, which are hereby incorporated in their
entireties by reference herein. Additionally, glutamine synthase expression
vectors can be
obtained from Lonza Biologics, Inc. (Portsmouth, NH). Expression and
production of
monoclonal antibodies using a GS expression system in murine myeloma cells is
described in
Bebbington et al., Bioltechr~ology 10:169(1992) and in Biblia and Robinson
Biotechnol.
Prog. 11:1 (1995) which are herein incorporated by reference.
[170] The present invention also relates to host cells containing the above-
described
vector constructs described herein, and additionally encompasses host cells
containing
nucleotide sequences of the invention that are operably associated with one or
mare
heterologous control regions (e.g., promoter and/or enhancer) using techniques
known of in
the art. The host cell can be a higher eukaryotic cell, such as a mammalian
cell (e.g., a
human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell can be a
prokaryotic cell, such as a bacterial cell. A host strain may be chosen which
modulates the
expression of the inserted gene sequences, or modifies and processes the gene
product in the
specific fashion desired. Expression from certain promoters can be elevated in
the presence
of certain inducers; thus expression of the genetically engineered polypeptide
may be
controlled. Furthermore, different host cells have characteristics and
specific mechanisms for
the translational and post-translational processing and modification (e.g.,
phosphorylation,
cleavage) of proteins. Appropriate cell lines can be chosen to ensure the
desired
modifications and processing of the foreign protein expressed.
[171] Introduction of the nucleic acids and nucleic acid constructs of the
invention into
the host cell can be effected by calcium phosphate transfection, DEAF-dextran
mediated
transfection, cationic lipid-mediated transfection, electroporation;
transduction, infection, or
other methods. Such methods are described in many standard laboratory manuals,
such as
Davis et al., Basic Methods In Molecular Biology (1986). It is specifically
contemplated that
the polypeptides of the present invention may in fact be expressed by a host
cell lacking a
recombinant vector.
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(172] In addition to encompassing host cells containing the vector constructs
discussed
herein, the invention also encompasses primary, secondary, and immortalized
host cells of
vertebrate origin, particularly mammalian origin, that have been engineered to
delete or
replace endogenous genetic material (e.g., the coding sequence), and/or to
include genetic
material (e.g., heterologous polynucleotide sequences) that is operably
associated with
polynucleotides of the invention, and which activates, alters, and/or
amplifies endogenous
polynucleotides. For example, techniques known in the art may be used to
operably associate
heterologous control regions (e.g., promoter and/or enhancer) and endogenous
polynucleotide
sequences via homologous recombination (see, e.g., US Patent Number 5,641,670,
issued
June 24, 1997; International Publication Number WO 96129411; International
Publication
Number WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); and
Zijlstra et al., Nature 3~?:435-438 (1989), the disclosures of each of which
are incorporated
by reference in their entireties).
[173] Polypeptides of the invention can be recovered and purified from
recombinant cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or canon exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography and lectin chromatography. Most preferably, high performance
liquid
chromatography ("HPLC") is employed for purification.
(174] Polypeptides of the present invention can also be recovered from:
products
purified from natural sources, including bodily fluids, tissues and cells,
whether directly
isolated or cultured; products of chemical synthetic procedures; and products
produced by
recombinant techniques from a prokaryotic or eukaryotic host, including, for
example,
bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon
the host
employed in a recombinant production procedure, the polypeptides of the
present invention
may be glycosylated or may be non-glycosylated. In addition, polypeptides of
the invention
may also include an initial modified methionine residue, in some cases as a
result of host-
mediated processes. Thus, it is well known in the art that the N-terminal
methionine encoded
by the translation initiation codon generally is removed with high efficiency
from any protein
after translation in all eukaryotic cells. While the N-terminal methionine on
most proteins
also is efficiently removed in most prokaryotes, for some proteins, this
prokaryotic removal
process is inefficient, depending on the nature of the amino acid to which the
N-terminal
methionine is covalently linked.
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(175] In one embodiment, the yeast Pichia pastoris is used to express
polypeptides of the
invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast
which can
metabolize methanol as its sole carbon source. A main step in the methanol
metabolization
pathway is the oxidation of methanol to formaldehyde using Oz. This reaction
is catalyzed
by the enzyme alcohol oxidase. In order to metabolize methanol as its sole
carbon source,
Pichia pastoris must generate high levels of alcohol oxidase due, in part, to
the relatively low
affinity of alcohol oxidase for OZ. Consequently, in a growth medium depending
on
methanol as a main carbon source, the promoter region of one of the two
alcohol oxidase
genes (AOXI) is highly active. In the presence of methanol, alcohol oxidase
produced from
the AOXI gene comprises up to approximately 30% of the total soluble protein
in Pichia
pastoris. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz,
P.J, et al., Yeast
5:167-77 (1989); Tschopp, J.F., et al., Nucl. Acids Res. 15:3859-76 (1987).
Thus, a
heterologous coding sequence, such as, for example, a polynucleotide of the
present
invention, under the transcriptional regulation of all or part of the AOXI
regulatory sequence
is expressed at exceptionally high levels in Pichia yeast grown in the
presence of methanol.
[176] In one example, the plasmid vector pPIC9K is used to express DNA
encoding a
polypeptide of the invention, as set forth herein, in a Pichea yeast system
essentially as
described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins
and J. Cregg,
eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows
expression and
secretion of a polypeptide of the invention by virtue of the strong AOXI
promoter linked to
the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e.,
leader) located
upstream of a multiple cloning site.
[177] Many other yeast vectors could be used in place of pPIC9K, such as,
pYES2,
pYDI, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2,
pHIL-S l, pPIC3.5K, and PA0815, as one skilled in the art would readily
appreciate, as long
as the proposed expression construct provides appropriately located signals
for transcription,
translation, secretion (if desired), and the like, including an in-frame AUG
as required.
[178] In another embodiment, high-level expression of a heterologous coding
sequence,
such as, for example, a polynucleotide of the present invention, may be
achieved by cloning
the heterologous polynucleotide of the invention into an expression vector
such as, for
example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of
methanol.
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[179] In addition to encompassing host cells containing the vector constructs
discussed
herein, the invention also encompasses primary, secondary, and immortalized
host cells of
vertebrate origin, particularly mammalian origin, that have been engineered to
delete or
replace endogenous genetic material (e.g., coding sequence), and/or to include
genetic
material (e.g., heterologous polynucleotide sequences) that is operably
associated with
polynucleotides of the invention, and which activates, alters, and/or
amplifies endogenous
polynucleotides. For example, techniques known in the art may be used to
operably associate
heterologous control regions (e.g., promoter and/or enhancer) and endogenous
polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent
No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/29411,
published
September 26, 1996; International Publication No. WO 94/12650, published
August 4, 1994;
Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et
al., Nature
342:435-438 (1989), the disclosures of each of which are incorporated by
reference in their
entireties).
[180] In addition, polypeptides of the invention can be chemically synthesized
using
techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures
and Molecular
Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-
111 (1984)).
For example, a polypeptide corresponding to a fragment of a polypeptide can be
synthesized
by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino
acids or
chemical amino acid analogs can be introduced as a substitution or addition
into the
polypeptide sequence. Non-classical amino acids include, but are not limited
to, to the D-
isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric
acid, 4-
aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic
acid, Aib,
2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-
butylglycine, t-
butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,
designer
amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl
amino acids,
and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L
(levorotary).
[181] The invention encompasses polypeptides of the present invention which
are
differentially modified during or after translation, e.g., by glycosylation,
acetylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any
of numerous
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chemical modifications may be earned out by known techniques, including but
not limited, to
specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain,
V8
protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic
synthesis in the
presence of tunicamycin; etc.
[182] Additional post-translational modifications encompassed by the invention
include,
for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-
terminal or
C-terminal ends), attachment of chemical moieties to the amino acid backbone,
chemical
modifications of N-linked or O-linked carbohydrate chains, and addition or
deletion of an
N-terminal methionine residue as a result of procaryotic host cell expression.
The
polypeptides may also be modified with a detectable label, such as an
enzymatic, fluorescent,
isotopic or affinity label to allow for detection and isolation of the
protein.
[183] Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic
group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent
materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material include
iodine ('z'I,'z3I,'zsl,'3~I), carbon ('4C), sulfur (3sS), tritium (3H), indium
("'In, "zIn, 113m~~
iism ~~ ~9m zoo 6g 6~ alladium '°3Pd
In), technetium ( Tc, Tc), thallium ( Ti), gallium ( Ga, Ga), p ( ),
molybdenum (99Mo), xenon ('33Xe), fluorine ('8F), 's3Sm, '~~Lu, 's9Gd, 'a9Pm,
l4oLa, l~sYb,
166H~~ ~01,~ 4~Sc~ ~s6Re~ issRe~ ~azPr~ ios~~ and 9~Ru.
[184] In specific embodiments, a polypeptide of the present invention or
fragment or
variant thereof is attached to macrocyclic chelators that associate with
radiometal ions,
including but not limited to, '~~Lu, 9°Y, 166Ho, and 's3Sm, to
polypeptides. In a preferred
embodiment, the radiometal ion associated with the macrocyclic chelators is
"'In. In
another preferred embodiment, the radiometal ion associated with the
macrocyclic chelator is
9°Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-
tetraazacyclododecane-
N,N',N",N"'-tetraacetic acid (DOTA). In other specific embodiments, DOTA is
attached to an
antibody of the invention or fragment thereof via a linker molecule. Examples
of linker
molecules useful for conjugating DOTA to a polypeptide are commonly known in
the art -
see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998);
Peterson et al.,
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Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol.
26(8):943-50
(1999); which are hereby incorporated by reference in their entirety.
[185J As mentioned, the proteins of the invention may be modified by either
natural
processes, such as posttranslational processing, or by chemical modification
techniques
which are well known in the art. It will be appreciated that the same type of
modification
may be present in the same or varying degrees at several sites in a given
polypeptide.
Polypeptides of the invention may be branched, for example, as a result of
ubiquitination, and
they may be cyclic, with or without branching. Cyclic, branched, and branched
cyclic
polypeptides may result from posttranslation natural processes or may be made
by synthetic
methods. Modifications include acetylation, acylation, ADP-ribosylation,
amidation,
covalent attachment of flavin, covalent attachment of a heme moiety, covalent
attachment of
a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid
derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine, formation
of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor
formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation,
proteolytic
processing, phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-
RNA mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.
Enzymol. 182:626-
646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[186] Also provided by the invention are chemically modified derivatives of
the
polypeptides of the invention which may provide additional advantages such as
increased
solubility, stability and circulating time of the polypeptide, or decreased
immunogenicity (see
U.S. Patent No. 4,179,337). The chemical moieties for derivitization may be
selected from
water soluble polymers such as polyethylene glycol, ethylene glycol/propylene
glycol
copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The
polypeptides may be modified at random positions within the molecule, or at
predetermined
positions within the molecule and may include one, two, three or more attached
chemical
moieties.
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[187] The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1 kDa
and about 100 kDa (the term "about" indicating that in preparations of
polyethylene glycol,
some molecules will weigh more, some less, than the stated molecular weight)
for ease in
handling and manufacturing. Other sizes may be used, depending on the desired
therapeutic
profile (e.g., the duration of sustained release desired, the effects, if any
on biological
activity, the ease in handling, the degree or lack of antigenicity and other
known effects of
the polyethylene glycol to a therapeutic protein or analog). For example, the
polyethylene
glycol may have an average molecular weight of about 200, 500, 1000, 1500,
2000, 2500,
3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,
9500,
10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000,
15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,
20,000, 25,000,
30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000,
75,000, 80,000,
85,000, 90,000, 95,000, or 100,000 kDa.
[188] As noted above, the polyethylene glycol may have a branched structure.
Branched
polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575;
Morpurgo et
al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides
18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999),
the
disclosures of each of which are incorporated herein by reference.
[189] The polyethylene glycol molecules (or other chemical moieties) should be
attached
to the protein with consideration of effects on functional or antigenic
domains of the protein.
There are a number of attachment methods available to those skilled in the
art, such as, for
example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein
incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035
(1992),
reporting pegylation of GM-CSF using tresyl chloride. For example,
polyethylene glycol
may be covalently bound through amino acid residues via a reactive group, such
as a free
amino or carboxyl group. Reactive groups are those to which an activated
polyethylene
glycol molecule may be bound. The amino acid residues having a free amino
group may
include lysine residues and the N-terminal amino acid residues; those having a
free carboxyl
group may include aspartic acid residues glutamic acid residues and the C-
terminal amino
acid residue. Sulfhydryl groups may also be used as a reactive group for
attaching the
polyethylene glycol molecules. Preferred for therapeutic purposes is
attachment at an amino
group, such as attachment at the N-terminus or lysine group.
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[190] As suggested above, polyethylene glycol may be attached to proteins via
linkage to
any of a number of amino acid residues. For example, polyethylene glycol can
be linked to
proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic
acid, or cysteine
residues. One or more reaction chemistries may be employed to attach
polyethylene glycol
to specific amino acid residues (e.g., lysine, histidine, aspartic acid,
glutamic acid, or
cysteine) of the protein or to more than one type of amino acid residue (e.g.,
lysine, histidine,
aspartic acid, glutamic acid, cysteine and combinations thereof) of the
protein.
[191] One may specifically desire proteins chemically modified at the N-
terminus.
Using polyethylene glycol as an illustration of the present composition, one
may select from
a variety of polyethylene glycol molecules (by molecular weight, branching,
etc.), the
proportion of polyethylene glycol molecules to protein (polypeptide) molecules
in the
reaction mix, the type of pegylation reaction to be performed, and the method
of obtaining
the selected N-terminally pegylated protein. The method of obtaining the N-
terminally
pegylated preparation (i.e., separating this moiety from other monopegylated
moieties if
necessary) may be by purification of the N-terminally pegylated material from
a population
of pegylated protein molecules. Selective proteins chemically modified at the
N-terminus
modification may be accomplished by reductive alkylation which exploits
differential
reactivity of different types of primary amino groups (lysine versus the N-
terminal) available
for derivatization in a particular protein. Under the appropriate reaction
conditions,
substantially selective derivatization of the protein at the N-terminus with a
carbonyl group
containing polymer is achieved.
[192] As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may be
attached
to the protein either directly or by an intervening linker. Linkerless systems
for attaching
polyethylene glycol to proteins are described in Delgado et al., Crit. Rev.
Thera. Drug Carrier
Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);
U.S. Patent No.
4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the
disclosures of
each of which are incorporated herein by reference.
[193] One system for attaching polyethylene glycol directly to amino acid
residues of
proteins without an intervening linker employs tresylated MPEG, which is
produced by the
modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride
(CISOzCH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene
glycol is
directly attached to amine groups of the protein. Thus, the invention includes
protein-
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polyethylene glycol conjugates produced by reacting proteins of the invention
with a
polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[194] Polyethylene glycol can also be attached to proteins using a number of
different
intervening linkers. For example, U.S. Patent No. 5,612,460, the entire
disclosure of which is
incorporated herein by reference, discloses urethane linkers for connecting
polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein the
polyethylene glycol is
attached to the protein by a linker can also be produced by reaction of
proteins with
compounds such as MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-
nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of
additional
polyethylene glycol derivatives and reaction chemistries for attaching
polyethylene glycol to
proteins are described in International Publication No. WO 98/32466, the
entire disclosure of
which is incorporated herein by reference. Pegylated protein products produced
using the
reaction chemistries set out herein are included within the scope of the
invention.
[195] The number of polyethylene glycol moieties attached to each protein of
the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated proteins
of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 15, 17, 20, or
more polyethylene glycol molecules. Similarly, the average degree of
substitution within
ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-
14, 13-15, 14-16,
15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein
molecule. Methods
for determining the degree of substitution are discussed, for example, in
Delgado et al., Crit.
Rev. Thera. Drug Carner Sys. 9:249-304 (1992).
[196] The polypeptides of the invention can be recovered and purified from
chemical
synthesis and recombinant cell cultures by standard methods which include, but
are not
limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation
exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction
chromatography, affinity chromatography, hydroxylapatite chromatography and
lectin
chromatography. Most preferably, high performance liquid chromatography
("HPLC") is
employed for purification. Well known techniques for refolding protein may be
employed to
regenerate active conformation when the polypeptide is denatured during
isolation and/or
purification.
(197] The polypeptides of the invention may be in monomers or multimers (i.e.,
dimers,
trimers, tetramers and higher multimers). Accordingly, the present invention
relates to
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monomers and multimers of the polypeptides of the invention, their
preparation, and
Compositions (preferably, Therapeutics) containing them. In specific
embodiments, the
polypeptides of the invention are monomers, dimers, trimers or tetramers. In
additional
embodiments, the multimers of the invention are at least dimers, at least
trimers, or at least
tetramers.
[198] Multimers encompassed by the invention may be homomers or heteromers. As
used herein, the term homomer refers to a multimer containing only
polypeptides
corresponding to a protein of the invention (e.g., the amino acid sequence of
SEQ ID NO:Y,
an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID
NO:X, the
amino acid sequence encoded by the portion of SEQ ID NO:X as defined in
columns 8 and 9
of Table 2, and/or an amino acid sequence encoded by cDNA contained in Clone
ID NO:Z
(including fragments, variants, splice variants, and fusion proteins,
corresponding to these as
described herein)). These homomers may contain polypeptides having identical
or different
amino acid sequences. In a specific embodiment, a homomer of the invention is
a multimer
containing only polypeptides having an identical amino acid sequence. In
another specific
embodiment, a homomer of the invention is a multimer containing polypeptides
having
different amino acid sequences. In specific embodiments, the multimer of the
invention is a
homodimer (e.g., containing two polypeptides having identical or different
amino acid
sequences) or a homotrimer (e.g., containing three polypeptides having
identical and/or
different amino acid sequences). In additional embodiments, the homomeric
multimer of the
invention is at least a homodimer, at least a homotrimer, or at least a
homotetramer.
(199] As used herein, the term heteromer refers to a multimer containing one
or more
heterologous polypeptides (i.e., polypeptides of different proteins) in
addition to the
polypeptides of the invention. In a specific embodiment, the multimer of the
invention is a
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the heteromeric
multimer of the invention is at least a heterodimer, at least a heterotrimer,
or at least a
heterotetramer.
[200] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic
and/or covalent associations and/or may be indirectly linked by, for example,
liposome
formation. Thus, in one embodiment, multimers of the invention, such as, for
example,
homodimers or homotrimers, are formed when polypeptides of the invention
contact one
another in solution. In another embodiment, heteromultimers of the invention,
such as, for
example, heterotrimers or heterotetramers, are formed when polypeptides of the
invention
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contact antibodies to the polypeptides of the invention (including antibodies
to the
heterologous polypeptide sequence in a fusion protein of the invention) in
solution. In other
embodiments, multimers of the invention are formed by covalent associations
with and/or
between the polypeptides of the invention. Such covalent associations may
involve one or
more amino acid residues contained in the polypeptide sequence (e.g., that
recited in SEQ m
NO:Y, encoded by the portion of SEQ m NO:X as defined in columns 8 and 9 of
Table 2,
and/or encoded by the cDNA contained in Clone ID NO:Z). In one instance, the
covalent
associations are cross-linking between cysteine residues located within the
polypeptide
sequences which interact in the native (i.e., naturally occurring)
polypeptide. In another
instance, the covalent associations are the consequence of chemical or
recombinant
manipulation. Alternatively, such covalent associations may involve one or
more amino acid
residues contained in the heterologous polypeptide sequence in a fusion
protein. In one
example, covalent associations are between the heterologous sequence contained
in a fusion
protein of the invention (see, e.g., US Patent Number 5,478,925). In a
specific example, the
covalent associations are between the heterologous sequence contained in a Fc
fusion protein
of the invention (as described herein). In another specific example, covalent
associations of
fusion proteins of the invention are between heterologous polypeptide sequence
from another
protein that is capable of forming covalently associated multimers, such as
for example,
osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the
contents of which
are herein incorporated by reference in its entirety). In another embodiment,
two or more
polypeptides of the invention are joined through peptide linkers. Examples
include those
peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by
reference).
Proteins comprising multiple polypeptides of the invention separated by
peptide linkers may
be produced using conventional recombinant DNA technology.
[201] Another method for preparing multimer polypeptides of the invention
involves use
of polypeptides of the invention fused to a leucine zipper or isoleucine
zipper polypeptide
sequence. Leucine zipper and isoleucine zipper domains are polypeptides that
promote
multimerization of the proteins in which they are found. Leucine zippers were
originally
identified in several DNA-binding proteins (Landschulz et al., Science
240:1759, (1988)),
and have since been found in a variety of different proteins. Among the known
leucine
zippers are naturally occurring peptides and derivatives thereof that dimerize
or trimerize.
Examples of leucine zipper domains suitable for producing soluble multimeric
proteins of the
invention are those described in PCT application WO 94/10308, hereby
incorporated by
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reference. Recombinant fusion proteins comprising a polypeptide of the
invention fused to a
polypeptide sequence that dimerizes or trimerizes in solution are expressed in
suitable host
cells, and the resulting soluble multimeric fusion protein is recovered from
the culture
supernatant using techniques known in the art.
[202] Trimeric polypeptides of the invention may offer the advantage of
enhanced
biological activity. Preferred leucine zipper moieties and isoleucine moieties
are those that
preferentially form trimers. One example is a leucine zipper derived from lung
surfactant
protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994))
and in U.S.
patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides
derived from naturally occurring trimeric proteins may be employed in
preparing trimeric
polypeptides of the invention.
[203] In another example, proteins of the invention are associated by
interactions
between Flag~ polypeptide sequence contained in fusion proteins of the
invention containing
Flag~ polypeptide sequence. In a further embodiment, proteins of the invention
are
associated by interactions between heterologous polypeptide sequence contained
in Flag~
fusion proteins of the invention and anti-Flag~ antibody.
[204] The multimers of the invention may be generated using chemical
techniques
known in the art. For example, polypeptides desired to be contained in the
multimers of the
invention may be chemically cross-linked using linker molecules and linker
molecule length
optimization techniques known in the art (see, e.g., US Patent Number
5,478,925, which is
herein incorporated by reference in its entirety). Additionally, multimers of
the invention
may be generated using techniques known in the art to form one or more inter-
molecule
cross-links between the cysteine residues located within the sequence of the
polypeptides
desired to be contained in the multimer (see, e.g., US Patent Number
5,478,925, which is
herein incorporated by reference in its entirety). Further, polypeptides of
the invention may
be routinely modified by the addition of cysteine or biotin to the C-terminus
or N-terminus of
the polypeptide and techniques known in the art may be applied to generate
multimers
containing one or more of these modified polypeptides (see, e.g., US Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally,
techniques known in the art may be applied to generate liposomes containing
the polypeptide
components desired to be contained in the multimer of the .invention (see,
e.g., US Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
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[205] Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, polypeptides
contained in
multimers of the invention are produced recombinantly using fusion protein
technology
described herein or otherwise known in the art (see, e.g., US Patent Number
5,478,925,
which is herein incorporated by reference in its entirety). In a specific
embodiment,
polynucleotides coding for a homodimer of the invention are generated by
ligating a
polynucleotide sequence encoding a polypeptide of the invention to a sequence
encoding a
linker polypeptide and then further to a synthetic polynucleotide encoding the
translated
product of the polypeptide in the reverse orientation from the original C-
terminus to the N-
terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925,
which is
herein incorporated by reference in its entirety). In another embodiment,
recombinant
techniques described herein or otherwise known in the art are applied to
generate
recombinant polypeptides of the invention which contain a transmembrane domain
(or
hydrophobic or signal peptide) and which can be incorporated by membrane
reconstitution
techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety).
Antibodies
[206] Further polypeptides of the invention relate to antibodies and T-cell
antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment, or
variant of the invention (e.g., a polypeptide or fragment or variant of the
amino acid sequence
of SEQ 117 NO:Y or a polypeptide encoded by the cDNA contained in Clone ID
No:Z, and/or
an epitope, of the present invention) as determined by immunoassays well known
in the art
for assaying specific antibody-antigen binding. Antibodies of the invention
include, but are
not limited to, polyclonal, monoclonal, multispecific, human, humanized or
chimeric
antibodies, single chain antibodies, Fab fragments, F(ab') fragments,
fragments produced by
a Fab expression library, anti-idiotypic (anti-Id) antibodies (including,
e.g., anti-Id antibodies
to antibodies of the invention), intracellularly-made antibodies (i.e.,
intrabodies), and
epitope-binding fragments of any of the above. The term "antibody," as used
herein, refers to
immunoglobulin molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site that
immunospecifically binds
an antigen. The immunoglobulin molecules of the invention can be of any type
(e.g., IgG,
IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI and
IgA2) or
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subclass of immunoglobulin molecule. In preferred embodiments, the
immunoglobulin
molecules of the invention are IgGI. In other preferred embodiments, the
immunoglobulin
molecules of the invention are IgG4.
(207] Most preferably the antibodies are human antigen-binding antibody
fragments of
the present invention and include, but are not limited to, Fab, Fab' and
F(ab')2, Fd, single-
chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and
fragments
comprising either a VL or VH domain. Antigen-binding antibody fragments,
including
single-chain antibodies, may comprise the variable regions) alone or in
combination with the
entirety or a portion of the .following: hinge region, CH1, CH2, and CH3
domains. Also
included in the invention are antigen-binding fragments also comprising any
combination of
variable regions) with a hinge region, CHI, CH2, and CH3 domains. The
antibodies of the
invention may be from any animal origin including birds and mammals.
Preferably, the
antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat,
guinea pig,
camel, horse, or chicken. As used herein, "human" antibodies include
antibodies having the
amino acid sequence of a human immunoglobulin and include antibodies isolated
from
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immunoglobulins, as
described infra
and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
[208] The antibodies of the present invention may be monospecific, bispecific,
trispecific
or of greater multispecificity. Multispecific antibodies may be specific for
different epitopes
of a polypeptide of the present invention or may be specific for both a
polypeptide of the
present invention as well as for a heterologous epitope, such as a
heterologous polypeptide or
solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802;
WO
91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent
Nos.
4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J.
Immunol.
148:1547-1553 (1992).
[209] Antibodies of the present invention may be described or specified in
terms of the
epitope(s) or portions) of a polypeptide of the present invention which they
recognize or
specifically bind. The epitope(s) or polypeptide portions) may be specified as
described
herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous
amino acid
residues, or listed in the Tables and Figures. Preferred epitopes of the
invention include the
predicted epitopes shown in column 7 of Table 1A, as well as polynucleotides
that encode
these epitopes. Antibodies which specifically bind any epitope or polypeptide
of the present
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invention may also be excluded. Therefore, the present invention includes
antibodies that
specifically bind polypeptides of the present invention, and allows for the
exclusion of the
same.
[210] Antibodies of the present invention may also be described or specified
in terms of
their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of
a polypeptide of the present invention are included. Antibodies that bind
polypeptides with
at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least
70%, at least 65%,
at least 60%, at least 55%, and at least 50% identity (as calculated using
methods known in
the art and described herein) to a polypeptide of the present invention are
also included in the
present invention. In specific embodiments, antibodies of the present
invention cross-react
with murine, rat and/or rabbit homologs of human proteins and the
corresponding epitopes
thereof. Antibodies that do not bind polypeptides with less than 95%, less
than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than
55%, and less than 50% identity (as calculated using methods known in the art
and described
herein) to a polypeptide of the present invention are also included in the
present invention.
In a specific embodiment, the above-described cross-reactivity is with respect
to any single
specific antigenic or immunogenic polypeptide, or combinations) of 2, 3, 4, 5,
or more of the
specific antigenic and/or immunogenic polypeptides disclosed herein. Further
included in the
present invention are antibodies which bind polypeptides encoded by
polynucleotides which
hybridize to a polynucleotide of the present invention under stringent
hybridization
conditions (as described herein). Antibodies of the present invention may also
be described
or specified in terms of their binding affinity to a polypeptide of the
invention. Preferred
binding affinities include those with a dissociation constant or Kd less than
5 X 10-Z M, 10-z
M, S X 10-3 M, 10-3 M, 5 X 10~ M, 10~ M, 5 X 10-5 M, 10-5 M, 5 X 10-6 M, 10-
6M, 5 X 10-~
M, 10' M, 5 X 10-g M, 10-g M, 5 X 10-9 M, 10-~ M, 5 X 10-' ° M, 10-'
° M, 5 X 10-" M, 10-"
M, 5 X 10-' Z M, 10-' 2 M, 5 X 10-13 M, 10~' 3 M, 5 X 10-14 M, 10-' 4 M, 5 X
10-15 M, or 10'' S M.
[211] The invention also provides antibodies that competitively inhibit
binding of an
antibody to an epitope of the invention as determined by any method known in
the art for
determining competitive binding, for example, the immunoassays described
herein. In
preferred embodiments, the antibody competitively inhibits binding to the
epitope by at least
95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at
least 60%, or at
least 50%.
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[212] Antibodies of the present invention may act as agonists or antagonists
of the
polypeptides of the present invention. For example, the present invention
includes antibodies
which disrupt the receptor/ligand interactions with the polypeptides of the
invention either
partially or fully. Preferably, antibodies of the present invention bind an
antigenic epitope
disclosed herein, or a portion thereof. The invention features both receptor-
specific
antibodies and ligand-specific antibodies. The invention also features
receptor-specific
antibodies which do not prevent ligand binding but prevent receptor
activation. Receptor
activation (i.e., signaling) may be determined by techniques described herein
or otherwise
known in the art. For example, receptor activation can be determined by
detecting the
phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its
substrate by
immunoprecipitation followed by western blot analysis (for example, as
described supra). In
specific embodiments, antibodies are provided that inhibit ligand activity or
receptor activity
by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at
least 70%, at least
60%, or at least 50% of the activity in absence of the antibody.
[213] The invention also features receptor-specific antibodies which both
prevent ligand
binding and receptor activation as well as antibodies that recognize the
receptor-ligand
complex, and, preferably, do not specifically recognize the unbound receptor
or the unbound
ligand. Likewise, included in the invention are neutralizing antibodies which
bind the ligand
and prevent binding of the ligand to the receptor, as well as antibodies which
bind the ligand,
thereby preventing receptor activation, but do not prevent the ligand from
binding the
receptor. Further included in the invention are antibodies which activate the
receptor. These
antibodies may act as receptor agonists, i.e., potentiate or activate either
all or a subset of the
biological activities of the ligand-mediated receptor activation, for example,
by inducing
dimerization of the receptor. The antibodies may be specified as agonists,
antagonists or
inverse agonists for biological activities comprising the specific biological
activities of the
peptides of the invention disclosed herein. The above antibody agonists can be
made using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998);
Zhu et al.,
Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179
(1998);
Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.
Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson
et al., J. Biol.
Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);
Muller
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et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20
(1996) (which
ire all incorporated by reference herein in their entireties).
[214] Antibodies of the present invention may be used, for example, to purify,
detect,
and target the polypeptides of the present invention, including both in vitro
and in vivo
diagnostic and therapeutic methods. For example, the antibodies have utility
in
immunoassays for qualitatively and quantitatively measuring levels of the
polypeptides of the
present invention in biological samples. See, e.g., Harlow et al., Antibodies:
A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); incorporated by
reference
herein in its entirety. .
[215] ~ As discussed in more detail below, the antibodies of the present
invention may be
used either alone or in combination with other compositions. The antibodies
may further be
recombinantly fused to a heterologous polypeptide at the N- or C-terminus or
chemically
conjugated (including covalent and non-covalent conjugations) to polypeptides
or other
compositions. For example, antibodies of the present invention may be
recombinantly fused
or conjugated to molecules useful as labels in detection assays and effector
molecules such as
heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT
publications WO
92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387;
the
disclosures of which are incorporated herein by reference in their entireties.
[216] The antibodies of the invention include derivatives that are modified,
i.e, by the
covalent attachment of any type of molecule to the antibody such that covalent
attachment
does not prevent the antibody from generating an anti-idiotypic response. For
example, but
not by way of limitation, the antibody derivatives include antibodies that
have been modified,
e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by
known protecting/blocking groups, proteolytic cleavage, linkage to a cellular
ligand or other
protein, etc. Any of numerous chemical modifications may be carried out by
known
techniques, including, but not limited to specific chemical cleavage,
acetylation, formylation,
metabolic synthesis of tunicamycin, etc. Additionally, the derivative may
contain one or
more non-classical amino acids.
[217] T'he antibodies of the present invention may be generated by any
suitable method
known in the art. Polyclonal antibodies to an antigen-of interest can be
produced by various
procedures well known in the art. For example, a polypeptide of the invention
can be
administered to various host animals including, but not limited to, rabbits,
mice, rats, etc. to
induce the production of sera containing polyclonal antibodies specific for
the antigen.
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Various adjuvants may be used to increase the immunological response,
depending on the
host species, and include but are not limited to, Freund's (complete and
incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebaeterium parvum. Such adjuvants are also well known in the art.
[218] Monoclonal antibodies can be prepared using a wide variety of techniques
known
in the art including the use of hybridoma, recombinant, and phage display
technologies, or a
combination thereof. For example, monoclonal antibodies can be produced using
hybridoma
techniques including those known in the art and taught, for example, in Harlow
et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988);
Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier,
N.Y., 1981) (said references incorporated by reference in their entireties).
The term
"monoclonal antibody" as used herein is not limited to antibodies produced
through
hybridoma technology. The term "monoclonal antibody" refers to an antibody
that is derived
from a single clone, including any eukaryotic, prokaryotic, or phage clone,
and not the
method by which it is produced.
[219] Methods for producing and screening for specific antibodies using
hybridoma
technology are routine and well known in the art and are discussed in detail
in the Examples.
In a non-limiting example, mice can be immunized with a polypeptide of the
invention or a
cell expressing such peptide. Once an immune response is detected, e.g.,
antibodies specific
for the antigen are detected in the mouse serum, the mouse spleen is harvested
and
splenocytes isolated. The splenocytes are then fused by well known techniques
to any
suitable myeloma cells, for example cells from cell line SP20 available from
the ATCC.
Hybridomas are selected and cloned by limited dilution. The hybridoma clones
are then
assayed by methods known in the art for cells that secrete antibodies capable
of binding a
polypeptide of the invention. Ascites fluid, which generally contains high
levels of
antibodies, can be generated by immunizing mice with positive hybridoma
clones.
[220] Accordingly, the present invention provides methods of generating
monoclonal
antibodies as well as antibodies produced by the method comprising culturing a
hybridoma
cell secreting an antibody of the invention wherein, preferably, the hybridoma
is generated by
fusing splenocytes isolated from a mouse immunized with an antigen of the
invention with
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myeloma cells and then screening the hybridomas resulting from the fusion for
hybridoma
Tones that secrete an antibody able to bind a polypeptide of the invention.
[221] Another well known method for producing both polyclonal and monoclonal
human
B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for
generating
EBV-transformed B cell lines are commonly known in the art, such as, for
example, the
protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan
et al., Eds.,
1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by
reference. The
source of B cells for transformation is commonly human peripheral blood, but B
cells for
transformation may also be derived from other sources including, but not
limited to, lymph
nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are
generally made into
single cell suspensions prior to EBV transformation. Additionally, steps may
be taken to
either physically remove or inactivate T cells (e.g., by treatment with
cyclosporin A) in B
cell-containing samples, because T cells from individuals seropositive for
anti-EBV
antibodies can suppress B cell immortalization by EBV.
[222] In general, the sample containing human B cells is innoculated with EBV,
and
cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of
the B95-8 cell
line (ATCC #VR-1492). Physical signs of EBV transformation can generally be
seen
towards the end of the 3-4 week culture period. By phase-contrast microscopy,
transformed
cells may appear large, clear, hairy and tend to aggregate in tight clusters
of cells. Initially,
EBV lines are generally polyclonal. However, over prolonged periods of cell
cultures, EBV
lines may become monoclonal or polyclonal as a result of the selective
outgrowth of
particular B cell clones. Alternatively, polyclonal EBV transformed lines may
be subcloned
(e.g., by limiting dilution culture) or fused with a suitable fusion partner
and plated at
limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners
for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-
Ag8.653),
heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and
human
cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present
invention
also provides a method of generating polyclonal or monoclonal human antibodies
against
polypeptides of the invention or fragments thereof, comprising EBV-
transformation of
human B cells.
[223] Antibody fragments which recognize specific epitopes may be generated by
known
techniques. For example, Fab and F(ab')2 fragments of the invention may be
produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain
(to
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produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2
fragments contain
the variable region, the light chain constant region and the CH1 domain of the
heavy chain.
[224] For example, the antibodies of the present invention can also be
generated using
various phage display methods known in the art. In phage display methods,
functional
antibody domains are displayed on the surface of phage particles which carry
the
polynucleotide sequences encoding them. In a particular embodiment, such phage
can be
utilized to display antigen binding domains expressed from a repertoire or
combinatorial
antibody library (e.g., human or murine). Phage expressing an antigen binding
domain that
binds the antigen of interest can be selected or identified with antigen,
e.g., using labeled
antigen or antigen bound or captured to a solid surface or bead. Phage used in
these methods
are typically filamentous phage including fd and M13 binding domains expressed
from phage
with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused
to either the
phage gene III or gene VIII protein. Examples of phage display methods that
can be used to
make the antibodies of the present invention include those disclosed in
Brinkman et al., J.
Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-
186
(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-
18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos.
5,698,426;
5,223;409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated
herein by reference in its entirety.
[225] As described in the above references, after phage selection, the
antibody coding
regions from the phage can be isolated and used to generate whole antibodies,
including
human antibodies, or any other desired antigen binding fragment, and expressed
in any
desired host, including mammalian cells, insect cells, plant cells, yeast, and
bacteria, e.g., as
described in detail below. For example, techniques to recombinantly produce
Fab, Fab' and
F(ab')2 fragments can also be employed using methods known in the art such as
those
disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques
12(6):864-869
(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science
240:1041-1043
( 1988) (said references incorporated by reference in their entireties).
[226] Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al.,
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Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and
Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo
use of
antibodies in humans and in vitro detection assays, it may be preferable to
use chimeric,
humanized, or human antibodies. A chimeric antibody is a molecule in which
different
portions of the antibody are derived from different animal species, such as
antibodies having
a variable region derived from a murine monoclonal antibody and a human
immunoglobulin
constant region. Methods for producing chimeric antibodies are known in the
art. See e.g.,
Mornson, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986);
Gillies et al.,
(1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567;
and
4,816397, which are incorporated herein by reference in their entirety.
Humanized
antibodies are antibody molecules from non-human species antibody that binds
the desired
antigen having one or more complementarity determining regions (CDRs) from the
non-
human species and a framework regions from a human immunoglobulin molecule.
Often,
framework residues in the human framework regions will be substituted with the
corresponding residue from the CDR donor antibody to alter, preferably
improve, antigen
binding. These framework substitutions are identified by methods well known in
the art, e.g.,
by modeling of the interactions of the CDR and framework residues to identify
framework
residues important for antigen binding and sequence comparison to identify
unusual
framework residues at particular positions. (See, e.g., Queen et al., U.S.
Patent No.
5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by
reference in their entireties.) Antibodies can be humanized using a variety of
techniques
known in the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO
91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing
(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991);
Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al.,
PNAS 91:969-973
(1994)), and chain shuffling (U.S. Patent No. 5,565,332).
[227] Completely human antibodies are particularly desirable for therapeutic
treatment
of human patients. Human antibodies can be made by a variety of methods known
in the art
including phage display methods described above using antibody libraries
derived from
human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and
4,716,111; and
PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein
by
reference in its entirety.
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[228] Human antibodies can also be produced using transgenic mice which are
incapable
bf expressing functional endogenous immunoglobulins, but which can express
human
immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene
complexes may be introduced randomly or by homologous recombination into mouse
embryonic stem cells. Alternatively, the human variable region, constant
region, and
diversity region may be introduced into mouse embryonic stem cells in addition
to the human
heavy and light chain genes. The mouse heavy and light chain immunoglobulin
genes may
be rendered non-functional separately or simultaneously with the introduction
of human
immunoglobulin loci by homologous recombination. In particular, homozygous
deletion of
the JH region prevents endogenous antibody production. The modified embryonic
stem cells
are expanded and microinjected into blastocysts to produce chimeric mice. The
chimeric
mice are then bred to produce homozygous offspring which express human
antibodies. The
transgenic mice are immunized in the normal fashion with a selected antigen,
e.g., all or a
portion of a polypeptide of the invention. Monoclonal antibodies directed
against the
antigen can be obtained from the immunized, transgenic mice using conventional
hybridoma
technology. The human immunoglobulin transgenes harbored by the transgenic
mice
rearrange during B cell differentiation, and subsequently undergo class
switching and
somatic mutation. Thus, using such a technique, it is possible to produce
therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology
for producing
human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995).
For a
detailed discussion of this technology for producing human antibodies and
human
monoclonal antibodies and protocols for producing such antibodies, see, e.g.,
PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European
Patent
No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016;
5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and
6,114,598, which are
incorporated by reference herein in their entirety. 'In addition, companies
such as Abgenix,
Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide
human
antibodies directed against a selected antigen using technology similar to
that described
above.
[229] Completely human antibodies which recognize a selected epitope can be
generated
using a technique referred to as "guided selection." In this approach a
selected non-human
monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of
a completely
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human antibody recognizing the same epitope. (Jespers et al., Biotechnology
12:899-903
(1988)).
[230] Further, antibodies to the polypeptides of the invention can, in turn,
be utilized to
generate anti-idiotype antibodies that "mimic" polypeptides of the invention
using~techniques
well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.
7(5):437-444;
(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,
antibodies
which bind to and competitively inhibit polypeptide multimerization and/or
binding of a
polypeptide of the invention to a ligand can be used to generate anti-
idiotypes that "mimic"
the polypeptide multimerization and/or binding domain and, as a consequence,
bind to and
neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of
such anti-idiotypes can be used in therapeutic regimens to neutralize
polypeptide
ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used
to bind a
polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and
thereby block its
biological activity. Alternatively, antibodies which bind to and enhance
polypeptide
multimerization and/or binding, and/or receptor/ligand multimerization,
binding and/or
signaling can be used to generate anti-idiotypes that function as agonists of
a polypeptide of
the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab
fragments of
such anti-idiotypes can be used in therapeutic regimens as agonists of the
polypeptides of the
invention or its ligand(s)/receptor(s). For example, such anti-idiotypic
antibodies can be used
to bind a polypeptide of the invention and/or to bind its
ligand(s)/receptor(s), and thereby
promote or enhance its biological activity.
[231] Intrabodies of the invention can be produced using methods known in the
art, such
as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601
(1994); Marasco,
W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol.
51:257-283
(1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene
17:2445-2456
(1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al., J.
Mol. Biol.
291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu
et al., J.
Immunol. Methods 231:207-222 (1999); and references cited therein.
Polynucleotides Encoding Antibodies
(232) The invention further provides polynucleotides comprising a nucleotide
sequence
encoding an antibody of the invention and fragments thereof. The invention
also
encompasses polynucleotides that hybridize under stringent or alternatively,
under lower
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stringency hybridization conditions, e.g., as defined supra, to
polynucleotides that encode an
antibody, preferably, that specifically binds to a polypeptide of the
invention, preferably, an
antibody that binds to a polypeptide having the amino acid sequence of SEQ ID
NO:Y, to a
polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9
of Table 2,
and/or to a polypeptide encoded by the cDNA contained in Clone ID NO:Z.
[233] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the nucleotide
sequence of the antibody is known, a polynucleotide encoding the antibody may
be
assembled from chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et
al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of
overlapping
oligonucleotides containing portions of the sequence encoding the antibody,
annealing and
ligating of those oligonucleotides, and then amplification of the ligated
oligonucleotides by
PCR.
[234] Alternatively, a polynucleotide encoding an antibody may be generated
from
nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding a particular
antibody is not available, but the sequence of the antibody molecule is known,
a nucleic acid
encoding the immunoglobulin may be chemically synthesized or obtained from a
suitable
source (e.g., an antibody cDNA library, or a cDNA library generated from, or
nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells expressing the
antibody, such as
hybridoma cells selected to express an antibody of the invention) by PCR
amplification
using synthetic primers hybridizable to the 3' and 5' ends of the sequence or
by cloning using
an oligonucleotide probe specific for the particular gene sequence to
identify, e.g., a cDNA
clone from a cDNA library that encodes the antibody. Amplified nucleic acids
generated by
PCR may then be cloned into replicable cloning vectors using any method well
known in the
art.
[235] Once the nucleotide sequence and corresponding amino acid sequence of
the
antibody is determined, the nucleotide sequence of the antibody may be
manipulated using
methods well known in the art for the manipulation of nucleotide sequences,
e.g.,
recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for
example, the
techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory
Manual, 2d
Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al.,
eds., 1998,
Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both
incorporated by reference herein in their entireties ), to generate antibodies
having a different
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amino acid sequence, for example to create amino acid substitutions,
deletions, and/or
insertions.
[236] In a specific embodiment, the amino aczd sequence of the heavy and/or
light chain
variable domains may be inspected to identify the sequences of the
complementarity
determining regions (CDRs) by methods that are well know in the art, e.g., by
comparison to
known amino acid sequences of other heavy and light chain variable regions to
determine the
regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or
more of the CDRs may be inserted within framework regions, e.g., into human
framework
regions to humanize a non-human antibody, as described supra. The framework
regions may
be naturally occurnng or consensus framework regions, and preferably human
framework
regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a
listing of human
framework regions). Preferably, the polynucleotide generated by the
combination of the
framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of
the invention. Preferably, as discussed supra, one or more amino acid
substitutions may be
made within the framework regions, and, preferably, the amino acid
substitutions improve
binding of the antibody to its antigen. Additionally, such methods may be used
to make
amino acid substitutions or deletions of one or more variable region cysteine
residues
participating in an intrachain disulfide bond to generate antibody molecules
lacking one or
more intrachain disulfide bonds. Other alterations to the polynucleotide are
encompassed by
the present invention and within the skill of the art.
[237] In addition, techniques developed for the production of "chimeric
antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al.,
Nature
312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing
genes from a
mouse antibody molecule of appropriate antigen specificity together with genes
from a
human antibody molecule of appropriate biological activity can be used. As
described supra,
a chimeric antibody is a molecule in which different portions are derived from
different
animal species, such as those having a variable region derived from a murine
mAb and a
human immunoglobulin constant region, e.g., humanized antibodies.
[238] Alternatively, techniques described for the production of single chain
antibodies
(U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988); Huston et al.,
Proc. Natl.
Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989))
can be
adapted to produce single chain antibodies. Single chain antibodies are formed
by linking
the heavy and light chain fragments of the Fv region via an amino acid bridge,
resulting in a
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single chain polypeptide. Techniques for the assembly of functional Fv
fragments in E. coli
may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Methods o, f Producing Antibodies
[239] The antibodies of the invention can be produced by any method known in
the art
for the synthesis of antibodies, in particular, by chemical synthesis or
preferably, by
recombinant expression techniques. Methods of producing antibodies include,
but are not
limited to, hybridoma technology, EBV transformation, and other methods
discussed herein
as well as through the use recombinant DNA technology, as discussed below.
[240] Recombinant expression of an antibody of the invention, or fragment,
derivative or
analog thereof, (e.g., a heavy or light chain of an antibody of the invention
or a single chain
antibody of the invention), requires construction of an expression vector
containing a
polynuclebtide that encodes the antibody. Once a polynucleotide encoding an
antibody
molecule or a heavy or light chain of an antibody, or portion thereof
(preferably containing
the heavy or light chain variable domain), of the invention has been obtained,
the vector for
the production of the antibody molecule may be produced by recombinant DNA
technology
using techniques well known in the art. Thus, methods for preparing a protein
by expressing
a polynucleotide containing an antibody encoding nucleotide sequence are
described herein.
Methods which are well known to those skilled in the art can be used to
construct expression
vectors containing antibody coding sequences and appropriate transcriptional
and
translational control signals. These methods include, for example, in vitro
recombinant DNA
techniques, synthetic techniques, and in vivo genetic recombination. The
invention, thus,
provides replicable vectors comprising a nucleotide sequence encoding an
antibody molecule
of the invention, or a heavy or light chain thereof, or a heavy or light chain
variable domain,
operably linked to a promoter. Such vectors may include the nucleotide
sequence encoding
the constant region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT
Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable
domain of the
antibody may be cloned into such a vector for expression of the entire heavy
or light chain.
[241] The expression vector is transferred to a host cell by conventional
techniques and
the transfected cells are then cultured by conventional techniques to produce
an antibody of
the invention. Thus, the invention includes host cells containing a
polynucleotide encoding
an antibody of the invention, or a heavy or light chain thereof, or a single
chain antibody of
the invention, operably linked to a heterologous promoter. In preferred
embodiments for the
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CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
expression of double-chained antibodies, vectors encoding both the heavy and
light chains
may be co-expressed in the host cell for expression of the entire
immunoglobulin molecule,
as detailed below.
[242] A variety of host-expression vector systems may be utilized to express
the
antibody molecules of the invention. Such host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but also
represent cells which may, when transformed or transfected with the
appropriate nucleotide
coding sequences, express an antibody molecule of the invention in situ. These
include but
are not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia)
transformed with
recombinant yeast expression vectors containing antibody coding sequences;
insect cell
systems infected with recombinant virus expression vectors (e.g., baculovirus)
containing
antibody coding sequences; plant cell systems infected with recombinant virus
expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed
with recombinant plasmid expression vectors (e.g., Ti plasmid) containing
antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells)
harboring
recombinant expression constructs containing promoters derived from the genome
of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,
bacterial cells
such as Escherichia coli, and more preferably, eukaryotic cells, especially
for the expression
of whole recombinant antibody molecule, are used for the expression of a
recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster ovary
cells
(CHO), in conjunction with a vector such as the major intermediate early gene
promoter
element from human cytomegalovirus is an effective expression system for
antibodies
(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2
(1990)).
[243] In bacterial systems, a number of expression vectors may be
advantageously
selected depending upon the use intended for the antibody molecule being
expressed. For
example, when a large quantity of such a protein is to be produced, for the
generation of
pharmaceutical compositions of an antibody molecule, vectors which direct the
expression
of high levels of fusion protein products that are readily purified may be
desirable. Such
vectors include, but are not limited, to the E. coli expression vector pUR278
(Ruther et al.,
EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated
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individually into the vector in frame with the lac Z coding region so that a
fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109
(1985); Van
Heeke & Schuster, J. Biol. Chem. 24:5503-5509.(1989)); and the like. pGEX
vectors may
also be used to express foreign polypeptides as fusion proteins with
glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily be purified
from lysed
cells by adsorption and binding to matrix glutathione-agarose beads followed
by elution in
the presence of free glutathione. The pGEX vectors are designed to include
thrombin or
factor Xa protease cleavage sites so that the cloned target gene product can
be released from
the GST moiety.
[244] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is
used as a vector to express foreign genes. The virus grows in Spodoptera
frugiperda cells.
The antibody coding sequence may be cloned individually into non-essential
regions (for
example the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter
(for example the polyhedrin promoter).
[245] In mammalian host cells, a number of viral-based expression systems may
be
utilized. In cases where an adenovirus is used as an expression vector, the
antibody coding
sequence of interest may be ligated to an adenovirus transcription/translation
control
complex, e.g., the late promoter and tripartite leader sequence. This chimeric
gene may then
be inserted in the adenovirus genome by in vitro or in vivo recombination.
Insertion in a non-
essential region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus
that is viable and capable of expressing the antibody molecule in infected
hosts. (e.g., see
Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific
initiation signals
may also be required for efficient translation of inserted antibody coding
sequences. These
signals include the ATG initiation codon and adjacent sequences. Furthermore,
the initiation
codon must be in phase with the reading frame of the desired coding sequence
to ensure
translation of the entire insert. These exogenous translational control
signals and initiation
codons can be of a variety of origins, both natural and synthetic. The
efficiency of
expression may be enhanced by the inclusion of appropriate transcription
enhancer elements,
transcription terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[246] In addition, a host cell strain may be chosen which modulates the
expression of the
inserted sequences, or modifies and processes the gene product in the specific
fashion
desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein
products may be important for the function of the protein. Different host
cells have
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characteristic and specific mechanisms for the post-translational processing
and modification
of proteins and gene products. Appropriate cell lines or host systems can be
chosen to
ensure the correct modification and processing of the foreign protein
expressed. To this end,
eukaryotic host cells which possess the cellular machinery for proper
processing of the
primary transcript, glycosylation, and phosphorylation of the gene product may
be used.
Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela,
COS,
MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for
example,
BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such
as, for
example, CRL7030 and Hs578Bst.
[247] For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. For example, cell lines which stably express the antibody molecule
may be
engineered. Rather than using expression vectors which contain viral origins
of replication,
host cells can be transformed with DNA controlled by appropriate expression
control
elements (e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation
sites, etc.), and a selectable marker. Following the introduction of the
foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched media, and
then are
switched to a selective media. The selectable marker in the recombinant
plasmid confers
resistance to the selection and allows cells to stably integrate the plasmid
into their
chromosomes and grow to form foci which in turn can be cloned and expanded
into cell
lines. This method may advantageously be used to engineer cell lines which
express the
antibody molecule. Such engineered cell lines may be particularly useful in
screening and
evaluation of compounds that interact directly or indirectly with the antibody
molecule.
[248] A number of selection systems may be used, including but not limited to
the herpes
simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA
48:202
(1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817
(1980)) genes can
be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite
resistance can be
used as the basis of selection for the following genes: dhfr, which confers
resistance to
methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et
al., Proc. Natl.
Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic
acid
(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which
confers
resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and
Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-
596 (1993);
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CA 02395398 2002-06-20
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Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.
Biochem.
62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which
confers
resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known
in the art of recombinant DNA technology may be routinely applied to select
the desired
recombinant clone, and such methods are described, for example, in Ausubel et
al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and
in Chapters
12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons,
NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are
incorporated by
reference herein in their entireties.
(249] The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on gene
amplification for the expression of cloned genes in mammalian cells in DNA
cloning, Vol.3.
(Academic Press, New York, 1987)). When a marker in the vector system
expressing
antibody is amplifiable, increase in the level of inhibitor present in culture
of host cell will
increase the number of copies of the marker gene. Since the amplified region
is associated
with the antibody gene, production of the antibody will also increase (Grouse
et al., Mol.
Cell. Biol. 3:257 (1983)).
[250] Vectors which use glutamine synthase (GS) or DHFR as the selectable
markers
can be amplified in the presence of the drugs methionine sulphoximine or
methotrexate,
respectively. An advantage of glutamine synthase based vectors are the
availabilty of cell
lines (e.g., the marine myeloma cell line, NSO) which are glutamine synthase
negative.
Glutamine synthase expression systems can also function in glutamine synthase
expressing
cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional
inhibitor to prevent
the functioning of ~ the endogenous gene. A glutamine synthase expression
system and
components thereof are detailed in PCT publications: W087/04462; W086/05807;
W089/01036; W089/10404; and W091/06657 which are incorporated in their
entireties by
reference herein. Additionally, glutamine synthase expression vectors that may
be used
according to the present invention are commercially available from suplliers,
including, for
example Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of
monoclonal
antibodies using a GS expression system in marine myeloma cells is described
in Bebbington
et al., Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechnol.
Prog. 11:1
(1995) which are incorporated in their entirities by reference herein.
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[251 ] The host cell may be co-transfected with two expression vectors of the
invention,
the first vector encoding a heavy chain derived polypeptide and the second
vector encoding a
light chain derived polypeptide. The two vectors may contain identical
selectable markers
which enable equal expression of heavy and light chain polypeptides.
Alternatively, a single
vector may be used which encodes, and is capable of expressing, both heavy and
light chain
polypeptides. In such situations, the light chain should be placed before the
heavy chain to
avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986);
Kohler, Proc.
Natl. Acad. Sci. USA 77:2197 (1980)). -The coding sequences for the heavy and
light chains
may comprise cDNA or genomic DNA.
[252] Once an antibody molecule of the invention has been produced by an
animal,
chemically synthesized, or recombinantly expressed, it may be purified by any
method
known in the art for purification of an immunoglobulin molecule, for example,
by
chromatography (e.g., ion exchange, affinity, particularly by affinity for the
specific antigen
after Protein A, and sizing column chromatography), centrifugation,
differential solubility, or
by any other standard technique for the purification of proteins. In addition,
the antibodies of
the present invention or fragments thereof can be fused to heterologous
polypeptide
sequences described herein or otherwise known in the art, to facilitate
purification.
[253] The present invention encompasses antibodies recombinantly fused or
chemically
conjugated (including both covalently and non-covalently conjugations) to a
polypeptide (or
portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100
amino acids of the
polypeptide) of the present invention to generate fusion proteins. The fusion
does not
necessarily need to be direct, but may occur through linker sequences. The
antibodies may
be specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20,
30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the
present invention.
For example, antibodies may be used to target the polypeptides of the present
invention to
particular cell types, either in vitro or in vivo, by fusing or conjugating
the polypeptides of
the present invention to antibodies specific for particular cell surface
receptors. Antibodies
fused or conjugated to the polypeptides of the present invention may also be
used in in vitro
immunoassays and purification methods using methods known in the art. See
e.g., Harbor et
al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al.,
Immunol. Lett.
39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432
(1992); Fell et
al., J. Immunol. 146:2446=2452 (1991), which are incorporated by reference in
their
entireties.
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[254] The present invention further includes compositions comprising the
polypeptides
of the present invention fused or conjugated to antibody domains other than
the variable
regions. For example, the polypeptides of the present invention may be fused
or conjugated
to an antibody Fc region, or portion thereof. The antibody portion fused to a
polypeptide of
the present invention may comprise the constant region, hinge region, CHl
domain, CH2
domain, and CH3 domain or any combination of whole domains or portions
thereof. The
polypeptides may also be fused or conjugated to the above antibody portions to
form
multimers. For example, Fc portions fused to the polypeptides of the present
invention can
form dimers through disulfide bonding between the Fc portions. Higher
multimeric forms
can be made by fusing the polypeptides to portions of IgA and IgM. Methods for
fusing or
conjugating the polypeptides of the present invention to antibody portions are
known in the
art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
5,447,851;
5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570;
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et
al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA
89:11337- 11341
(1992) (said references incorporated by reference in their entireties).
[255J As discussed, supra, the polypeptides corresponding to a polypeptide,
polypeptide
fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above
antibody
portions to increase the in vivo half life of the polypeptides or for use in
immunoassays using
methods known in the art. Further, the polypeptides corresponding to SEQ ID
NO:Y may be
fused or conjugated to the above antibody portions to facilitate purification.
One reported
example describes chimeric proteins consisting of the first two domains of the
human CD4-
polypeptide and various domains of the constant regions of the heavy or light
chains of
mammalian immunoglobulins. See EP 394,827; and Traunecker et al., Nature
331:84-86
(1988). The polypeptides of the present invention fused or conjugated to an
antibody having
disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in binding
and neutralizing other molecules, than the monomeric secreted protein or
protein fragment
alone. See, for example, Fountoulakis et al., J. Biochem. 270:3958-3964
(1995). In many
cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis,
and thus can result
in, for example, improved pharmacokinetic properties. See, for example, EP A
232,262.
Alternatively, deleting the Fc part after the fusion protein has been
expressed, detected, and
purified, would be desired. 'For example, the Fc portion may hinder therapy
and diagnosis if
the fusion protein is used as an antigen for immunizations. In drug discovery,
for example,
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human proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-
throughput screening assays to identify antagonists of hIL-5. (See, Bennett et
al., J.
Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-
9471
(1995)).
[256] Moreover, the antibodies or fragments thereof of the present invention
can be
fused to marker sequences, such as a peptide to facilitate purification. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311),
among others, many of which are commercially available. As described in Gentz
et al., Proc.
Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides
for
convenient purification of the fusion protein. Other peptide tags useful for
purification
include, but are not limited to, the "HA" tag, which corresponds to an epitope
derived from
the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and
the "flag" tag.
[257] The present invention further encompasses antibodies or fragments
thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically
to, for example, monitor the development or progression of a tumor as part of
a clinical
testing procedure to, e.g., determine the efficacy of a given treatment
regimen. Detection
can be facilitated by coupling the antibody to a detectable substance.
Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent
materials, bioluminescent materials, radioactive materials, positron emitting
metals using
various positron emission tomographies, and nonradioactive paramagnetic metal
ions. The
detectable substance may be coupled or conjugated either directly to the
antibody (or
fragment thereof) or indirectly, through an intermediate (such as, for
example, a linker known
in the art) using techniques known in the art. See, for example, U.S. Patent
No. 4,741,900 for
metal ions which can be conjugated to antibodies for use as diagnostics
according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic
group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent
materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material include
125I, 131I, 1 llln or 99Tc.
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[258] Further, an antibody or fragment thereof may be conjugated to a
therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or a
radioactive metal ion, e.g., alpha-emitters such , as, for example, 213Bi. A
cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells. Examples
include paclitaxol, '
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione,
mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-
mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating agents
(e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and
lomustine
(CCN~, cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin
C, and
cis- dichlorodiamine platinum (In (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin .
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents
(e.g., vincristine and vinblastine).
[259] The conjugates of the invention can be used for modifying a given
biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to classical
chemical therapeutic agents. For example, the drug moiety may be a protein or
polypeptide
possessing a desired biological activity. Such proteins may include, for
example, a toxin
such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein
such as tumor
necrosis factor, a-interferon, 13-interferon, nerve growth factor, platelet
derived growth factor,
tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta,
AIM I (See,
International Publication No. WO 97/33899), AIM II (See, International
Publication No. WO
97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)),
VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or an anti-
angiogenic
agent, e.g., angiostatin or endostatin; or, biological response modifiers such
as, for example,
lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6
("IL,-6"),
granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte
colony
stimulating factor ("G-CSF"), or other growth factors.
[260] Antibodies may also be attached to solid supports, which are
particularly useful for
immunoassays or purification of the target antigen. Such solid supports
include, but are not
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limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or
polypropylene.
[261] Techniques for conjugating such therapeutic moiety to antibodies are
well known.
See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In
Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp.
243-56 (Alan R. Liss, Inc. 1.985); Hellstrom et al., "Antibodies For Drug
Delivery", in
Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel
Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et
al. (eds.), pp.
475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic
Use Of
Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer
Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press
1985), and Thorpe
et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin
Conjugates",
Immunol. Rev. 62:119-58 (1982).
(262] Alternatively, an antibody can be conjugated to a second antibody to
form an
antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980,
which is
incorporated herein by reference in its entirety.
[263] An antibody, with or without a therapeutic moiety conjugated to it,
administered
alone or in combination with cytotoxic factors) and/or cytokine(s) can be used
as a
therapeutic.
Immunophenotyping
[264] The antibodies of the invention may be utilized for immunophenotyping of
cell
lines and biological samples. Translation products of the gene of the present
invention may
be useful as cell-specific markers, or more specifically as cellular markers
that are
differentially expressed at various stages of differentiation and/or
maturation of particular
cell types. Monoclonal antibodies directed against a specific epitope, or
combination of
epitopes, will allow for the screening of cellular populations expressing the
marker. Various
techniques can be utilized using monoclonal antibodies to screen for cellular
populations
expressing the marker(s), and include magnetic separation using antibody-
coated magnetic
beads, "panning" with antibody attached to a solid matrix (i.e., plate), and
flow cytometry
(See, e.g., U.S. Patent 5,985;660; and Morrison et al., Cell, 96:737-49
(1999)).
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[265] These techniques allow for the screening of particular populations of
cells, such as
might be found with hematological malignancies (i.e. minimal residual disease
(MRD) in
acute leukemic patients) and "non-self' cells in transplantations to prevent
Graft-versus-Host
Disease (GVHD). Alternatively, these techniques allow for the screening of
hematopoietic
stem and progenitor cells capable of undergoing proliferation and/or
differentiation, as might
be found in human umbilical cord blood.
Assays For Antibody Binding
[266] The antibodies of the invention may be assayed for immunospecific
binding by
any method known in the art. The immunoassays which can be used include but
are not
limited to competitive and non-competitive assay systems using techniques such
as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin
reactions, irrimunodiffusion assays, agglutination assays, complement-fixation
assays,
immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays, to
name but a few. Such assays are routine and well known in the art (see, e.g.,
Ausubel et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons,
Inc., New
York, which is incorporated by reference herein in its entirety). Exemplary
immunoassays
are described briefly below (but are not intended by way of limitation).
(267] Immunoprecipitation protocols generally comprise lysing a population of
cells in a
lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium
deoxycholate,
0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol)
supplemented
with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium
vanadate), adding the antibody of interest to the cell lysate, incubating for
a period of time
(e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose
beads to the cell lysate,
incubating for about an hour or more at 4° C, washing the beads in
lysis buffer and
resuspending the beads in SDS/sample buffer. The ability of the antibody of
interest to
immunoprecipitate a particular antigen can be assessed by, e.g., western blot
analysis. One
of skill in the art would be knowledgeable as to the parameters that can be
modified to
increase the binding of the antibody to an antigen and decrease the background
(e.g., pre-
clearing the cell lysate with sepharose beads). For further discussion
regarding
immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
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[268] Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein
sample from the
polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon,
blocking the
membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing
the
membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with
primary
antibody (the antibody of interest) diluted in blocking buffer, washing the
membrane in
washing buffer, blocking the membrane with a secondary antibody (which
recognizes the
primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic
substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 1251)
diluted in blocking buffer, washing the membrane in wash buffer, and detecting
the presence
of the antigen. One of skill in the art would be knowledgeable as to the
parameters that can
be modified to increase the signal detected and to reduce the background
noise. For further
discussion regarding western blot protocols see, e.g., Ausubel et al, eds,
(1994), Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York,
section 10.8.1.
[269] ELISAs comprise preparing antigen, coating the well of a 96 well
microtiter plate
with the antigen, adding the antibody of interest conjugated to a detectable
compound such
as an enzymatic substrate (e.g., horseradish peroxidase or alkaline
phosphatase) to the well
and incubating for a period of time, and detecting the presence of the
antigen. In ELISAs the
antibody of interest does not have to be conjugated to a detectable compound;
instead, a
second antibody (which recognizes the antibody of interest) conjugated to a
detectable
compound may be added to the well. Further, instead of coating the well with
the antigen,
the antibody may be coated to the well. In this case, a second antibody
conjugated to a
detectable compound may be added following the addition of the antigen of
interest to the
coated well. One of skill in the art would be knowledgeable as to the
parameters that can be


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affinity of the antibody of interest for a particular antigen and the binding
off rates can be
determined from the data by scatchard plot analysis. Competition with a second
antibody
can also be determined using radioimmunoassays. In this case, the antigen is
incubated with
antibody of interest conjugated to a labeled compound (e.g., 3H or 1251) in
the presence of
increasing amounts of an unlabeled second antibody.
(271] Antibodies of the invention may be characterized using
immunocytochemisty
methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a
vector
enabling the expression of an antigen or with vector alone using techniques
commonly
known in the art. Antibodies that bind antigen transfected cells, but not
vector-only
transfected cells, are antigen specific.
Therapeutic Uses
[272] The present invention is further directed to antibody-based therapies
which involve
administering antibodies of the invention to an animal, preferably a mammal,
and most
preferably a human, patient for treating one or more of the disclosed
diseases, disorders, or
conditions. Therapeutic compounds of the invention include, but are not
limited to,
antibodies of the invention (including fragments, analogs and derivatives
thereof as described
herein) and nucleic acids encoding antibodies of the invention (including
fragments, analogs
and derivatives thereof and anti-idiotypic antibodies as described herein).
The antibodies of
the invention can be used to treat, inhibit or prevent diseases, disorders or
conditions
associated with aberrant expression and/or activity of a polypeptide of the
invention,
including, but not limited to, any one or more of the diseases, disorders, or
conditions
described herein. The treatment and/or prevention of diseases, disorders, or
conditions
associated with aberrant expression and/or activity of a polypeptide of the
invention
includes, but is not limited to, alleviating symptoms associated with those
diseases, disorders
or conditions. Antibodies of the invention may be provided in pharmaceutically
acceptable
compositions as known in the art or as described herein.
(273] In a specific and preferred embodiment, the present invention is
directed to
antibody-based therapies which involve administering antibodies of the
invention to an
animal, preferably a mammal, and most preferably a human, patient for treating
one or more
diseases, disorders, or conditions, including but not limited to: neural
disorders, immune
system disorders, muscular disorders, reproductive disorders, gastrointestinal
disorders,
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pulmonary disorders, cardiovascular disorders, renal disorders, proliferative
disorders, and/or
cancerous diseases and conditions., and/or as described elsewhere herein.
Therapeutic
compounds of the invention include, but are not limited to, antibodies of the
invention (e.g.,
antibodies directed to the full length protein expressed on the cell surface
of a mammalian
cell; antibodies directed to an epitope of a polypeptide of the invention
(such as, for example,
a predicted linear epitope shown in column 7 of Table 1A; or a conformational
epitope,
including fragments, analogs and derivatives thereof as described herein) and
nucleic acids
encoding antibodies of the invention (including fragments, analogs and
derivatives thereof
and anti-idiotypic antibodies as described herein). The antibodies of the
invention can be
used to treat, inhibit or prevent diseases, disorders or conditions associated
with aberrant
expression and/or activity of a polypeptide of the invention, including, but
not limited to, any
one or more of the diseases, disorders, or conditions described herein. The
treatment and/or
prevention of diseases, disorders, or conditions associated with aberrant
expression and/or
activity of a polypeptide of the invention includes, but is not limited to,
alleviating symptoms
associated with those diseases, disorders or conditions. Antibodies of the
invention may be
provided in pharmaceutically acceptable compositions as known in the art or as
described
herein.
[274] A summary of the ways in which the antibodies of the present invention
may be
used therapeutically includes binding polynucleotides or polypeptides of the
present
invention locally or systemically in the body or by direct cytotoxicity of the
antibody, e.g. as
mediated by complement (CDC) or by effector cells (ADCC). Some of these
approaches are
described in more detail below. Armed with the teachings provided herein, one
of ordinary
skill in the art will know how to use the antibodies of the present invention
for diagnostic,
monitoring or therapeutic purposes without undue experimentation.
[275] The antibodies of this invention may be advantageously utilized in
combination
with other monoclonal or chimeric antibodies, or with lymphokines or
hematopoietic growth
factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number
or activity of effector cells which interact with the antibodies.
[276] The antibodies of the invention may be administered alone or in
combination with
other types of treatments (e.g., radiation therapy, chemotherapy, hormonal
therapy,
immunotherapy and anti-tumor agents). Generally, administration of products of
a species
origin or species reactivity (in the case of antibodies) that is the same
species as that of the
patient is preferred. Thus, in a preferred embodiment, human antibodies,
fragments
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derivatives, analogs, or nucleic acids, are administered to a human patient
for therapy or
prophylaxis.
[277] It is preferred to use high affinity , and/or potent in vivo inhibiting
and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention,
fragments or regions thereof, for both immunoassays directed to and therapy of
disorders
related to polynucleotides or polypeptides, including fragments thereof, of
the present
invention. Such antibodies, fragments, or regions, will preferably have an
affinity for
polynucleotides or polypeptides of the invention, including fragments thereof.
Preferred
binding affinities include those with a dissociation constant or Kd less than
5 X 10'Z M, 10'2
M, 5 X 10'3 M, 10'3 M, 5 X 10~ M, 10'~ M, 5 X 10'5 M, 10'5 M, 5 X 10'6 M, 10'6
M, 5 X 10'
M, 10-' M, 5 X 10'$ M, 10-g M, S X 10'9 M, 10'9 M, 5 X 10'' ° M, 10''
° M, 5 X 10''' M, 10-"
M, 5 X 10'' Z M, 10-' 2 M, 5 X 10'' 3 M, 10-' 3 M, 5 X 10-' 4 M, 10'' 4 M, S X
10'' S M, and 10''s
M.
Gene Therapy
[278] In a specific embodiment, nucleic acids comprising sequences encoding
antibodies
or functional derivatives thereof, are administered to treat, inhibit or
prevent a disease or
disorder associated with aberrant expression and/or activity of a polypeptide
of the invention,
by way of gene therapy. Gene therapy refers to therapy performed by the
administration to a
subject of an expressed or expressible nucleic acid. In this embodiment of the
invention, the
nucleic acids produce their encoded protein that mediates a therapeutic
effect.
[279] Any of the methods for gene therapy available in the art can be used
according to
the present invention. Exemplary methods are described below.
[280] For general reviews of the methods of gene therapy, see Goldspiel et
al., Clinical
Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev,
Ann.
Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932
(1993); and
Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH
11(5):155-
215 (1993). Methods commonly known in the art of recombinant DNA technology
which can
be used are described in Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A
Laboratory
Manual, Stockton Press, NY (1990).
[281] In a preferred embodiment, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors that
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express the antibody or fragments or chimeric proteins or heavy or light
chains thereof in a
suitable host. In particular, such nucleic acid sequences have promoters
operably linked to
the antibody coding region, said promoter being, inducible or constitutive,
and, optionally,
tissue-specific. In another particular embodiment, nucleic acid molecules are
used in which
the antibody coding sequences and any other desired sequences are flanked by
regions that
promote homologous recombination at a desired site in the genome, thus
providing for
intrachromosomal expression of the antibody encoding nucleic acids (Koller and
Smithies,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature
342:435-438 (1989).
In specific embodiments, the expressed antibody molecule is a single chain
antibody;
alternatively, the nucleic acid sequences include sequences encoding both the
heavy and
light chains, or fragments thereof, of the antibody.
[282] Delivery of the nucleic acids into a patient may be either direct, in
which case the
patient is directly exposed to the nucleic acid or nucleic acid- carrying
vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in vitro, then
transplanted into
the patient. These two approaches are known, respectively, as in vivo or ex
vivo gene
therapy.
[283] In a specific embodiment, the nucleic acid sequences are directly
administered in
vivo, where it is expressed to produce the encoded product. This can be
accomplished by
any of numerous methods known in the art, e.g., by constructing them as part
of an
appropriate nucleic acid expression vector and administering it so that they
become
intracellular, e.g., by infection using defective or attenuated retrovirals or
other viral vectors
(see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by
use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids or
cell-surface receptors or transfecting agents, encapsulation in liposomes,
microparticles, or
microcapsules, or by administering them in linkage to a peptide which is known
to enter the
nucleus, by administering it in linkage to a ligand subject to receptor-
mediated endocytosis
(see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used
to target
cell types specifically expressing the receptors), etc. In another embodiment,
nucleic acid
ligand complexes can be formed in which the ligand comprises a fusogenic viral
peptide to
disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
In yet another
embodiment, the nucleic acid can be targeted in vivo for cell specific uptake
and expression,
by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO
92/22635;
W092/20316; W093/14188, WO 93/20221). Alternatively, the nucleic acid can be
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introduced intracellularly and incorporated within host cell DNA for
expression, by
homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935
(1989); Zijlstra et al., Nature 342:435-438 (1989)).
[284] In a specific embodiment, viral vectors that contains nucleic acid
sequences
encoding an antibody of the invention are used. For example, a retroviral
vector can be used
(see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral
vectors contain the
components necessary for the correct packaging of the viral genome and
integration into the
host cell DNA. The nucleic acid sequences encoding the antibody to be used in
gene therapy
are cloned into one or more vectors, which facilitates delivery of the gene
into a patient.
More detail about retroviral vectors can be found in Boesen et al., Biotherapy
6:291-302
(1994), which describes the use of a retroviral vector to deliver the mdrl
gene to
hematopoietic stem cells in order to make the stem cells more resistant to
chemotherapy.
Other references illustrating the use of retroviral vectors in gene therapy
are: Clowes et al., J.
Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994);
Salmons and
Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr.
Opin.
in Genetics and Devel. 3:110-114 (1993).
[285] Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory epithelia.
Adenoviruses naturally infect respiratory epithelia where they cause a mild
disease. Other
targets for adenovirus-based delivery systems are liver, the central nervous
system,
endothelial cells, and muscle. Adenoviruses have the advantage of being
capable of infecting
non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development
3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et
al., Human
Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to
transfer genes to
the respiratory epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in
gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991);
Rosenfeld et al.,
Cell 68:143- 155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234
(1993); PCT
Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a
preferred
embodiment, adenovirus vectors are used.
[286] Adeno-associated virus (AAV) has also been proposed for use in gene
therapy
(Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
[287] Another approach to gene therapy involves transferring a gene to cells
in tissue
culture by such methods as electroporation, lipofection, calcium phosphate
mediated
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transfection, or viral infection. Usually, the method of transfer includes the
transfer of a
selectable marker to the cells. The cells are then placed under selection to
isolate those cells
that have taken up and are expressing the transferred gene. Those cells are
then delivered to
a patient.
[288] In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be carried out
by any method known in the art, including but not limited to transfection,
electroporation,
microinjection, infection with a viral or bacteriophage vector containing the
nucleic acid
sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated
gene
transfer, spheroplast fusion, etc. Numerous techniques are known in the art
for the
introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth.
Enzymol.
217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline,
Pharmac.
Ther. 29:69-92m (1985) and may be used in accordance with the present
invention, provided
that the necessary developmental and physiological functions of the recipient
cells are not
disrupted. The technique should provide for the stable transfer of the nucleic
acid to the cell,
so that the nucleic acid is expressible by the cell and preferably heritable
and expressible by
its cell progeny.
[289] The resulting recombinant cells can be delivered to a patient by various
methods
known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are
preferably administered intravenously. The amount of cells envisioned for use
depends on
the desired effect, patient state, etc., and can be determined by one skilled
in the art.
[290] Cells into which a nucleic acid can be introduced for purposes of gene
therapy
encompass any desired, available cell type, and include but are not limited to
epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes;
blood cells such as T
lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
megakaryocytes, granulocytes; various stem or progenitor cells, in particular
hematopoietic
stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord
blood,
peripheral blood, fetal liver, etc.
[291] In a preferred embodiment, the cell used for gene therapy is autologous
to the
patient.
[292] In an embodiment in which recombinant cells are used in gene therapy,
nucleic
acid sequences encoding am antibody are introduced into the cells such that
they are
expressible by the cells or their progeny, and the recombinant cells are then
administered in
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vivo for therapeutic effect. In a specific embodiment, stem or progenitor
cells are used. Any
stem and/or progenitor cells which can be isolated and maintained in vitro can
potentially be
used in accordance with this embodiment of the present invention (see e.g. PCT
Publication
WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth.
Cell Bio.
21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
[293] In a specific embodiment, the nucleic acid to be introduced for purposes
of gene
therapy comprises an inducible promoter operably linked to the coding region,
such that
expression of the nucleic acid is controllable by the presence or absence of
an appropriate
inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
[294] The compounds or pharmaceutical compositions of the invention are
preferably
tested in vitro, and then in vivo for the desired therapeutic or prophylactic
activity, prior to
use in humans. For example, in vitro assays to demonstrate the therapeutic or
prophylactic
utility of a compound or pharmaceutical composition include, the effect of a
compound on a
cell line or a patient tissue sample. The effect of the compound or
composition on the cell
line and/or tissue sample can be determined utilizing techniques known to
those of skill in the
art including, but not limited to, rosette formation assays and cell lysis
assays. In accordance
with the invention, in vitro assays which can be used to determine whether
administration of
a specific compound is indicated, include in vitro cell culture assays in
which a patient tissue
sample is grown in culture, and exposed to or otherwise, administered a
compound, and the
effect of such compound upon the tissue sample is observed.
TherapeuticlProphylactic Administration and Composition
[295] The invention provides methods of treatment, inhibition and prophylaxis
by
administration to a subject of an effective amount of a compound or
pharmaceutical
composition of the' invention, preferably a polypeptide or antibody of the
invention. In a
preferred embodiment, the compound is substantially purified (e.g.,
substantially free from
substances that limit its effect or produce undesired side-effects). The
subject is preferably
an animal, including but not limited to animals such as cows, pigs, horses,
chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably human.
[296] Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional
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appropriate formulations and routes of administration can be selected from
among those
described herein below.
[297] Various delivery systems are known and can be used to administer a
compound of
the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant
cells capable of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and
Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as
part of a
retroviral or other vector, etc. Methods of introduction include but are not
limited to
intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural,
and oral routes. The compounds or compositions may be administered by any
convenient
route, for example by infusion or bolus injection, by absorption through
epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.)
and may be
administered together with other biologically active agents. Administration
can be systemic
or local. In addition, it may be desirable to introduce the pharmaceutical
compounds or
compositions of the invention into the central nervous system by any suitable
route,
including intraventricular and intrathecal injection; intraventricular
injection may be
facilitated by an intraventricular catheter, for example, attached to a
reservoir, such as an
Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use
of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
[298] In a specific embodiment, it may be desirable to administer the
pharmaceutical
compounds or compositions of the invention locally to the area in need of
treatment; this may
be achieved by, for example, and not by way of limitation, local infusion
during surgery,
topical application, e.g., in conjunction with a wound dressing after surgery,
by injection, by
means of a catheter, by means of a suppository, or by means of an implant,
said implant
being of a porous, non-porous, or gelatinous material, including membranes,
such as sialastic
membranes, or fibers. Preferably, when administering a protein, including an
antibody, of
the invention, care must be taken to use materials to which the protein does
not absorb.
[299] In another embodiment, the compound or composition can be delivered in a
vesicle, in particular a liposome (see Larger, Science 249:1527-1533 (1990);
Treat et al., in
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler
(eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-
327; see
generally ibid.)
[300] In yet another embodiment, the compound or composition can be delivered
in a
controlled release system. In one embodiment, a pump may be used (see Larger,
supra;
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Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980);
Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric
materials can be used (see Medical Applications of Controlled Release, Langer
and Wise
(eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug
Bioavailability, Drug
Product Design and Performance, Smolen and Ball (eds.), Wiley, New York
(1984); Ranger
and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also
Levy et al.,
Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et
al.,
J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release
system can be
placed in proximity of the therapeutic target, e.g., the brain, thus requiring
only a fraction of
the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra,
vol. 2, pp. 115-138 (1984)).
[301] Other controlled release systems are discussed in the review by Langer
(Science
249:1527-1533 (1990)).
[302] In a specific embodiment where the compound of the invention is a
nucleic acid
encoding a protein, the nucleic acid can be administered in vivo to promote
expression of its
encoded protein, by constructing it as part of an appropriate nucleic acid
expression vector
and administering it so that it becomes intracellular, e.g., by use of a
retroviral vector (see
U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle
bombardment
(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface
receptors or
transfecting agents, or by administering it in linkage to a homeobox- like
peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.
USA 88:1864-1868
(1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly
and incorporated
within host cell DNA for expression, by homologous recombination.
[303] The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or a state
government or
listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for
use in
animals, and more particularly in humans. The term "carrier" refers to a
diluent, adjuvant,
excipient, or vehicle with which the therapeutic is administered. Such
pharmaceutical
Garners can be sterile liquids, such as water and oils, including those of
petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the
like. Water is a preferred carrier when the pharmaceutical composition is
administered
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intravenously. Saline solutions and aqueous dextrose and glycerol solutions
can also be
employed as liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if desired,
can also contain
minor amounts of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules,
powders, sustained-release formulations and the like. The composition can be
formulated as
a suppository, with traditional binders and carriers such as triglycerides.
Oral formulation
can include standard carriers such as pharmaceutical grades of mannitol,
lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of
suitable pharmaceutical carriers are described in "Remington's Pharmaceutical
Sciences" by
E.W. Martin. Such compositions will contain a therapeutically effective amount
of the
compound, preferably in purified form, together, with a suitable amount of
carrier so as to
provide the form for proper administration to the patient. The formulation
should suit the
mode of administration.
[304] In a preferred embodiment, the composition is formulated in accordance
with
routine procedures as a pharmaceutical composition adapted for intravenous
administration
to human beings. Typically, compositions for intravenous administration are
solutions in
sterile isotonic aqueous buffer. Where necessary, the composition may also
include a
solubilizing agent and a local anesthetic such as lignocaine to ease pain at
the site of the
injection. Generally, the ingredients are supplied either separately or mixed
together in unit
dosage form, for example, as a dry lyophilized powder or water free
concentrate in a
hermetically sealed container such as an ampoule or sachette indicating the
quantity of active
agent. Where the composition is to be administered by infusion, it can be
dispensed with an
infusion bottle containing sterile pharmaceutical grade water or saline. Where
the
composition is administered by injection, an ampoule of sterile water for
injection or saline
can be provided so that the ingredients may be mixed prior to administration.
[305] The compounds of the invention can be formulated as neutral or salt
forms.
Pharmaceutically acceptable salts include those formed with anions such as
those derived
from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with
cations such as those derived from sodium, potassium, ammonium, calcium,
ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
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[306] The amount of the compound of the invention which will be effective in
the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
expression and/or activity of a polypeptide of the invention can be determined
by standard
clinical techniques. In addition, in vitro assays may optionally be employed
to help identify
optimal dosage ranges. The precise dose to be employed in the formulation will
also depend
on the route of administration, and the seriousness of the disease or
disorder, and should be
decided according to the judgment of the practitioner and each patient's
circumstances.
Effective doses may be extrapolated from dose-response curves derived from in
vitro or
animal model test systems.
[307] For antibodies, the dosage administered to a patient is typically 0.1
mg/kg to 100
mg/kg of the patient's body weight. Preferably, the dosage administered to a
patient is
between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to
mg/kg of the patient's body weight. Generally, human antibodies have a longer
half life
within the human body than antibodies from other species due to the immune
response to the
foreign polypeptides. Thus, lower dosages of human antibodies and less
frequent
administration is often possible. Further, the dosage and frequency of
administration of
antibodies of the invention may be reduced by enhancing uptake and tissue
penetration (e.g.,
into the brain) of the antibodies by modifications such as, for example,
lipidation.
[308] The invention also provides a pharmaceutical pack or kit comprising one
or more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of
the invention. Optionally associated with such containers) can be a notice in
the form
prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency of
manufacture, use or sale for human administration.
Diagnosis and Imaging
[309] Labeled antibodies, and derivatives and analogs thereof, which
specifically bind to
a polypeptide of interest can be used for diagnostic purposes to detect,
diagnose, or monitor
diseases, disorders, and/or conditions associated with the aberrant expression
and/or activity
of a polypeptide of the invention. The invention provides for the detection of
aberrant
expression of a polypeptide of interest, comprising (a) assaying the
expression of the
polypeptide of interest in cells or body fluid of an individual using one or
more antibodies
specific to the polypeptide interest and (b) comparing the level of gene
expression with a
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CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
standard gene expression level, whereby an increase or decrease in the assayed
polypeptide
gene expression level compared to the standard expression level is indicative
of aberrant
expression.
[310] The invention provides a diagnostic assay for diagnosing a disorder,
comprising
(a) assaying the expression of the polypeptide of interest in cells or body
fluid of an
individual using one or more antibodies specific to the polypeptide interest
and (b)
comparing the level of gene expression with a standard gene expression level,
whereby an
increase or decrease in the assayed polypeptide gene expression level compared
to the
standard expression level is indicative of a particular disorder. With respect
to cancer, the
presence of a relatively high amount of transcript in biopsied tissue from an
individual may
indicate a predisposition for the development of the disease, or may provide a
means for
detecting the disease prior to the appearance of actual clinical symptoms. A
more definitive
diagnosis of this type may allow health professionals to employ preventative
measures or
aggressive treatment earlier thereby preventing the development or further
progression of the
cancer.
[311] Antibodies of the invention can be used to assay protein levels in a
biological
sample using classical immunohistological methods known to those of skill in
the art (e.g.,
see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J.
Cell . Biol. 105:3087-
3096 (1987)). Other antibody-based methods useful for detecting protein gene
expression
include immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in the art
and include
enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I,
1211), carbon
(14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);
luminescent labels,
such as luminol; and fluorescent labels, such as fluorescein and rhodamine,
and biotin.
[312] One facet of the invention is the detection and diagnosis of a disease
or disorder
associated with aberrant expression of a polypeptide of interest in an animal,
preferably a
mammal and most preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject
an effective amount of a labeled molecule which specifically binds to the
polypeptide of
interest; b) waiting for a time interval following the administering for
permitting the labeled
molecule to preferentially concentrate at sites in the subject where the
polypeptide is
expressed (and for unbound labeled molecule to be cleared to background
level); c)
determining background level; and d) detecting the labeled molecule in the
subject, such that
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CA 02395398 2002-06-20
WO 01/55163 PCT/USO1/01358
detection of labeled molecule above the background level indicates that the
subject has a
particular disease or disorder associated with aberrant expression of the
polypeptide of
interest. Background level can be determined by, various methods including,
comparing the
amount of labeled molecule detected to a standard value previously determined
for a
particular system.
[313] It will be understood in the art that the size of the subject and the
imaging system
used will determine the quantity of imaging moiety needed to produce
diagnostic images. In
the case of a radioisotope moiety, for a human subject, the quantity of
radioactivity injected
will normally range from about 5 to 20 millicuries of 99mTc. The labeled
antibody or
antibody fragment will then preferentially accumulate at the location of cells
which contain
the specific protein. In vivo tumor imaging is described in S.W. Burchiel et
al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments."
(Chapter 13
in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B.
A.
Rhodes, eds., Masson Publishing Inc. (1982)).
[314j Depending on several variables, including the type of label used and the
mode of
administration, the time interval following the administration for permitting
the labeled
molecule to preferentially concentrate at sites in the subject and for unbound
labeled
molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours
or 6 to 12 hours.
In another embodiment the time interval following administration is 5 to 20
days or 5 to 10
days.
[315] In an embodiment, monitoring of the disease or disorder is carried out
by repeating
the method for diagnosing the disease or disease, for example, one month after
initial
diagnosis, six months after initial diagnosis, one year after initial
diagnosis, etc.
(316] Presence of the labeled molecule can be detected in the patient using
methods
known in the art for in vivo scanning. These methods depend upon the type of
label used.
Skilled artisans will be able to determine the appropriate method for
detecting a particular
label. Methods and devices that may be used in the diagnostic methods of the
invention
include, but are not limited to, computed tomography (CT), whole body scan
such as position
emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
[317] In a specific embodiment, the molecule is labeled with a radioisotope
and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al., U.S.
Patent No. 5,441,050). In another embodiment, the molecule is labeled with a
fluorescent
compound and is detected in the patient using a fluorescence responsive
scanning instrument.
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In another embodiment, the molecule is labeled with a positron emitting metal
and is detected
in the patent using positron emission-tomography. 1n yet another embodiment,
the molecule
is labeled with a paramagnetic label and is detected in a patient using
magnetic resonance
imaging (MRI).
Kits
[318] The present invention provides kits that can be used in the above
methods. In one
embodiment, a kit comprises an antibody of the invention, preferably a
purified antibody, in
one or more containers. In a specific embodiment, the kits of the present
invention contain a
substantially isolated polypeptide comprising an epitope which is specifically
immunoreactive with an antibody included in the kit. Preferably, the kits of
the present
invention further comprise a control antibody which does not react with the
polypeptide of
interest. In another specific embodiment, the kits of the present invention
contain a means
for detecting the binding of an antibody to a polypeptide of interest (e.g.,
the antibody may be
conjugated to a detectable substrate such as a fluorescent compound, an
enzymatic substrate,
a radioactive compound or a luminescent compound, or a second antibody which
recognizes
the first antibody may be conjugated to a detectable substrate).
[319] In another specific embodiment of the present invention, the kit is a
diagnostic kit
for use in screening serum containing antibodies specific against
proliferative and/or
cancerous polynucleotides and polypeptides. Such a kit may include a control
antibody that
does not react with the polypeptide of interest. Such a kit may include a
substantially
isolated polypeptide antigen comprising an epitope which is specifically
immunoreactive
with at least one anti-polypeptide antigen antibody. Further, such a kit
includes means for
detecting the binding of said antibody to the antigen (e.g., the antibody may
be conjugated to
a fluorescent compound such as fluorescein or rhodamine which can be detected
by flow
cytometry). In specific embodiments, the kit may include a recombinantly
produced or
chemically synthesized polypeptide antigen. The polypeptide antigen of the kit
may also be
attached to a solid support.
[320] In a more specific embodiment the detecting means of the above-described
kit
includes a solid support to which said polypeptide antigen is attached. Such a
kit may also
include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of
the antibody to the polypeptide antigen can be detected by binding of the said
reporter-
labeled antibody.
379




DEMANDE OU BREVET VOLUMINEUX
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PLUS D'UN TOME.
CECI EST LE TOME 1 DE 3
~~ TTENANT LES PAGES 1 A 379
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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2001-01-17
(87) PCT Publication Date 2001-08-02
(85) National Entry 2002-06-20
Withdrawn Application 2002-11-20

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2002-06-20
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
ROSEN, CRAIG A.
BARASH, STEVEN C.
RUBEN, STEVEN M.
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 2002-06-20 381 15,333
Description 2002-06-20 33 1,352
Description 2002-06-20 267 15,327
Abstract 2002-06-20 2 169
Claims 2002-06-20 4 148
Cover Page 2002-11-22 2 111
PCT 2002-06-20 8 407
Assignment 2002-06-20 3 91
Correspondence 2002-11-19 1 24
Correspondence 2002-11-19 1 11
PCT 2002-06-20 1 85
Correspondence 2002-11-20 1 31
Correspondence 2002-12-11 1 14
PCT 2002-06-21 2 116
PCT 2002-06-21 7 296
PCT 2002-06-20 2 167
Assignment 2009-08-10 20 998